Screening assays using intramitochondrial calcium

ABSTRACT

The invention provides methods for screening for agents that modulate mitochondrial function and in particular mitochondrial regulation of intracellular calcium. The methods may be used to detect agents that bind to a mitochondrial calcium uniporter and may also detect inhibitors or uncouplers of mitochondrial respiration. Agents identified using the screens provided herein have application in the prevention and treatment of a variety of diseases associated with abnormal mitochondrial function.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/765,104, filed Jan. 16, 2001, issued Oct. 26, 2004, as U.S. Pat. No.6,808,873; which claims the benefit of U.S. Provisional Application No.60/176,384 filed Jan. 14, 2000, which are incorporated herein byreference in their entireties.

TECHNICAL FIELD

The invention relates generally to assays for screening for agents thataffect mitochondrial activity. More specifically, the invention isdirected to screening methods for use in identifying agents that altermitochondrial regulation of intracellular calcium. An assay for thepresence of extramitochondrial calcium and for factors that influencelevels of intramitochondrial and/or extramitochondrial calcium, such asthe calcium uniporter (CaUP), is provided herein.

BACKGROUND OF THE INVENTION

Mitochondria are organelles that are the main energy source in cells ofhigher organisms. These organelles provide direct and indirectbiochemical regulation of a wide array of cellular respiratory,oxidative and metabolic processes, including metabolic energyproduction, aerobic respiration and intracellular calcium regulation.For example, mitochondria are the site of electron transport chain (ETC)activity, which drives oxidative phosphorylation to produce metabolicenergy in the form of adenosine triphosphate (ATP), and which alsounderlies a central mitochondrial role in intracellular calciumhomeostasis. These processes require the maintenance of a mitochondrialmembrane electrochemical potential, and defects in such membranepotential can result in a variety of disorders.

In addition to their role in energy production in growing cells,mitochondria (or at least mitochondrial components) participate inprogrammed cell death (PCD), also known as apoptosis (see Newmeyer etal., Cell 79:353-364, 1994; Liu et al., Cell 86:147-157, 1996).Apoptosis is apparently required for normal development of the nervoussystem and functioning of the immune system. Some disease states areassociated with insufficient apoptosis (e.g., cancer and autoimmunediseases) or excessive levels of apoptosis (e.g., stroke andneurodegeneration). For general reviews of apoptosis, and the role ofmitochondria therein, see Green and Reed, Science 281:1309-1312, 1998;Green, Cell 94:695-698, 1998; and Kromer, Nature Medicine 3:614-620,1997.

Mitochondria contain an outer mitochondrial membrane that serves as aninterface between the organelle and the cytosol, a highly folded innermitochondrial membrane that appears to form attachments to the outermembrane at multiple sites, and an intermembrane space between the twomitochondrial membranes. The subcompartment within the innermitochondrial membrane is commonly referred to as the mitochondrialmatrix (for review, see, e.g., Emster et al., J. Cell Biol. 91:227s,1981). While the outer membrane is freely permeable to ionic andnon-ionic solutes having molecular weights less than about tenkilodaltons, the inner mitochondrial membrane exhibits selective andregulated permeability for many small molecules, including certaincations, and is impermeable to large (greater than about 10 kD)molecules.

Four of the five multisubunit protein complexes (Complexes I, III, IVand V) that mediate ETC activity are localized to the innermitochondrial membrane. The remaining ETC complex (Complex II) issituated in the matrix. In at least three distinct chemical reactionsknown to take place within the ETC, protons are moved from themitochondrial matrix, across the inner membrane, to the intermembranespace. This disequilibrium of charged species creates an electrochemicalmembrane potential of approximately 220 mV referred to as the“protonmotive force” (PMF). The PMF, which is often represented by thenotation Δp, corresponds to the sum of the electric potential (Δψm) andthe pH differential (ΔpH) across the inner membrane according to theequationΔp=Δψm−ZΔpHwherein Z stands for −2.303 RT/F. The value of Z is −59 at 25° C. whenΔp and Δψm are expressed in mV and ΔpH is expressed in pH units (see,e.g., Ernster et al., J. Cell Biol. 91:227s, 1981, and references citedtherein).

Δψm provides the energy for phosphorylation of adenosine diphosphate(ADP) to yield ATP by ETC Complex V, a process that is coupledstoichiometrically with transport of a proton into the matrix. Δψm isalso the driving force for the influx of cytosolic Ca²⁺ into themitochondrion. Under normal metabolic conditions, the inner membrane islargely impermeable to proton movement from the intermembrane space intothe matrix, leaving ETC Complex V as the primary means whereby protonscan return to the matrix. When, however, the integrity of the innermitochondrial membrane is compromised, as occurs during mitochondrialpermeability transition (MPT) that accompanies certain diseasesassociated with altered mitochondrial function, protons are able tobypass the conduit of Complex V without generating ATP, therebyuncoupling respiration (i.e., ETC activity) from ATP production. DuringMPT, Δψm collapses and mitochondrial membranes lose the ability toselectively regulate permeability to solutes both small (e.g., ionicCa²⁺, Na⁺, K⁺ and H⁺) and large (e.g., proteins). Loss of mitochondrialpotential also appears to be a critical event in the progression ofdiseases associated with altered mitochondrial function, includingdegenerative diseases such as Alzheimer's Disease; diabetes mellitus;Parkinson's Disease; Huntington's disease; dystonia; Leber's hereditaryoptic neuropathy; schizophrenia; mitochondrial encephalopathy, lacticacidosis, and stroke (MELAS); cancer; psoriasis; hyperproliferativedisorders; mitochondrial diabetes and deafness (MIDD) and myoclonicepilepsy ragged red fiber syndrome.

Normal alterations of intramitochondrial Ca²⁺ are associated with normalmetabolic regulation (Dykens, 1998 in Mitochondria & Free Radicals inNeurodegenerative Diseases, Beal, Howell and Bodis-Wollner, Eds.,Wiley-Liss, New York, pp. 29-55; Radi et al., 1998 in Mitochondria &Free Radicals in Neurodegenerative Diseases, Beal, Howell andBodis-Wollner, Eds., Wiley-Liss, New York, pp. 57-89; Gunter andPfeiffer, 1991, Am. J. Physiol. 27: C755; Gunter et al., Am. J. Physiol.267:313, 1994). For example, fluctuating levels of mitochondrial freeCa²⁺ may be responsible for regulating oxidative metabolism in responseto increased ATP utilization, via allosteric regulation of enzymes(reviewed by Crompton and Andreeva, Basic Res. Cardiol. 88:513-523,1993); and the glycerophosphate shuttle (Gunter and Gunter, J. Bioenerg.Biomembr. 26:471, 1994).

Normal mitochondrial function includes regulation of cytosolic freecalcium levels by sequestration of excess Ca²⁺ within the mitochondrialmatrix. Depending on cell type, cytosolic Ca²⁺ concentration istypically 50-100 nM. In normally functioning cells, when Ca²⁺ levelsreach 200-300 nM, mitochondria begin to accumulate Ca²⁺ as a function ofthe equilibrium between influx via a Ca²⁺ uniporter in the innermitochondrial membrane and Ca²⁺ efflux via both Na⁺ dependent and Na⁺independent calcium carriers. The low affinity of this rapid uniportermechanism suggests that the primary uniporter function may be to lowercytosolic Ca²⁺ in response to pathological elevation of cytosolic freecalcium levels, which may result from ATP depletion and/or abnormalcalcium influx across the plasma membrane (Gunter and Gunter, J.Bioenerg. Biomembr. 26:471, 1994; Gunter et al., Am. J. Physiol.267:313, 1994). In certain instances, such perturbation of intracellularcalcium homeostasis is a feature of diseases associated with alteredmitochondrial function, regardless of whether the calcium regulatorydysfunction is causative of, or a consequence of, altered mitochondrialfunction including MPT.

In view of the significance of mitochondrial regulation of intracellularcalcium and the relationship of this mitochondrial activity to severaldisease states, there is clearly a need for improved compositions andmethods to control mitochondrial calcium homeostasis. To provideimproved therapies for such diseases, agents that alter mitochondrialcalcium regulation may be beneficial, and assays to specifically detectsuch agents are needed. The present invention fulfills these needs andfurther provides other related advantages.

SUMMARY OF THE INVENTION

The present invention is directed in part to methods for identifyingagents that alter mitochondrial regulation of intracellular calcium.Thus, in one aspect the invention provides a method of identifying anagent that alters mitochondrial function, comprising (a) contacting, ineach of a plurality of reaction vessels in a high throughput screeningarray, (i)a biological sample comprising a cell containing cytosol, amitochondrion and a calcium indicator molecule, under conditions thatpermit maintenance of mitochondrial membrane potential, with (ii) asource of calcium cations, wherein the calcium indicator molecule iscapable of generating a detectable signal that is proportional to thelevel of calcium in the cytosol; (b) detecting in each reaction vesselthe signal generated by the calcium indicator molecule at a plurality oftime points; and (c) comparing the signal generated by the calciumindicator molecule at one or more of the time points in the absence of acandidate agent, to the signal generated by the calcium indicatormolecule at one or more of the time points in the presence of thecandidate agent, and therefrom identifying an agent that altersmitochondrial function. In one embodiment the step of contacting isrepeated at least once. In another embodiment the sample contains atleast one compound that alters intracellular distribution of a calciumcation. In a further embodiment the compound that alters intracellularcalcium cation distribution is thapsigargin or Ru360. In anotherembodiment the compound that alters intracellular calcium cationdistribution is a calcium ionophore or a membrane permeable compoundthat alters intracellular calcium distribution. In another embodimentthe sample contains at least one compound that uncouples oxidativephosphorylation from ATP production. In another embodiment the candidateagent is membrane permeable, and in another embodiment the calciumindicator molecule is membrane permeable. In another embodiment thesource of calcium cations is exogenous to the cell. In anotherembodiment the sample contains at least one compound that uncouplesoxidative phosphorylation from ATP production. In another embodiment thecell comprises at least one polypeptide that is a member of the Bcl-2family. In another embodiment the cell expresses a gene encoding apolypeptide that regulates cytosolic calcium. In another embodiment thegene encodes a mitochondrial calcium uniporter. In another embodimentthe gene is a transfected gene. In another embodiment the gene encodes amitochondrial calcium uniporter. In another embodiment the cell is apermeabilized cell. In certain embodiments the cell adheres to a solidsubstrate and in certain other embodiments the cell is a non-adherentcell.

It is another aspect of the present invention to provide a method ofidentifying an agent that uncouples oxidative phosphorylation from ATPproduction, comprising (a) contacting, in each of a plurality ofreaction vessels in a high throughput screening array, (i) a biologicalsample comprising a cell containing cytosol, a mitochondrion and acalcium indicator molecule, under conditions that permit maintenance ofmitochondrial membrane potential, with (ii) a source of calcium cations,wherein the calcium indicator molecule is capable of generating adetectable signal that is proportional to the level of calcium in thecytosol; (b) detecting in each reaction vessel the signal generated bythe calcium indicator molecule at a plurality of time points; (c)repeating steps (a) and (b) at least once; and (d) comparing (i) thesignal generated by the calcium indicator molecule at one or more of thetime points prior to and following at least one of the contacting stepsin the absence of the candidate agent, to (ii) the signal generated bythe calcium indicator molecule at one or more of the time points priorto and following at least one of the contacting steps in the presence ofthe candidate agent, wherein an increased level of calcium in thecytosol at a time point prior to a contacting step in the presence ofthe agent, compared to the level of calcium in the cytosol prior to acontacting step in the absence of the agent, indicates an agent thatuncouples oxidative phosphorylation from ATP production.

In another aspect the invention provides a method of identifying anagent that that is a respiratory inhibitor, comprising: (a) contacting,in each of a plurality of reaction vessels in a high throughputscreening array, (i) a biological sample comprising a cell containingcytosol, a mitochondrion and a calcium indicator molecule, underconditions that permit maintenance of mitochondrial membrane potential,with (ii)a source of calcium cations, wherein the calcium indicatormolecule is capable of generating a detectable signal that isproportional to the level of calcium in the cytosol; (b) detecting ineach reaction vessel the signal generated by the calcium indicatormolecule at a plurality of time points; (c) repeating steps (a) and (b)at least once; and (d) comparing (i) the signal generated by the calciumindicator molecule at one or more of the time points prior to andfollowing at least one of the contacting steps in the absence of thecandidate agent, to (ii) the signal generated by the calcium indicatormolecule at one or more of the time points prior to and following atleast one of the contacting steps in the presence of the candidateagent, wherein an increased level of calcium in the cytosol at a timepoint prior to a contacting step in the presence of the agent, comparedto the level of calcium in the cytosol prior to a contacting step in theabsence of the agent, indicates an agent that is a respiratoryinhibitor.

In another embodiment the invention provides a method of identifying anagent that alters a mitochondrial calcium uniporter, comprising: (a)contacting, in each of a plurality of reaction vessels in a highthroughput screening array, (i) a biological sample comprising a cellcontaining cytosol, a mitochondrion and a calcium indicator molecule,under conditions that permit maintenance of mitochondrial membranepotential, with (ii) a source of calcium cations, wherein the calciumindicator molecule is capable of generating a detectable signal that isproportional to the level of calcium in the cytosol; (b) detecting ineach reaction vessel the signal generated by the calcium indicatormolecule at a plurality of time points; (c) repeating steps (a) and (b)at least once; and (d) comparing (i) the signal generated by the calciumindicator molecule at one or more of the time points prior to andfollowing at least one of the contacting steps in the absence of thecandidate agent, to (ii) the signal generated by the calcium indicatormolecule at one or more of the time points prior to and following atleast one of the contacting steps in the presence of the candidateagent, wherein an increased level of calcium in the cytosol at a timepoint following a contacting step in the presence of the agent, comparedto the level of calcium in the cytosol following a contacting step inthe absence of the agent, indicates that the agent alters amitochondrial calcium uniporter.

In another embodiment there is provided a method of identifying an agentthat alters mitochondrial function, comprising (a) contacting (i) abiological sample comprising a cell containing cytosol, a mitochondrionand a calcium indicator molecule, under conditions that permitmaintenance of mitochondrial membrane potential, with (ii) a source ofcalcium cations, wherein the calcium indicator molecule is capable ofgenerating a detectable signal that is proportional to the level ofcalcium in the cytosol; (b) detecting the signal generated by thecalcium indicator molecule at a plurality of time points; and (c)comparing the signal generated by the calcium indicator molecule at oneor more of the time points in the absence of the candidate agent, to thesignal generated by the calcium indicator molecule at one or more of thetime points in the presence of the candidate agent, and therefromidentifying an agent that alters mitochondrial function. In oneembodiment the step of contacting is repeated at least once. In anotherembodiment the sample contains at least one compound that altersintracellular distribution of a calcium cation. In another embodimentthe compound that alters intracellular calcium cation distribution isthapsigargin or Ru360. In another embodiment the compound that altersintracellular calcium cation distribution is selected from the groupconsisting of a calcium ionophore and a membrane permeable compound thatalters intracellular calcium distribution. In another embodiment thesample contains at least one compound that uncouples oxidativephosphorylation from ATP production. In another embodiment the candidateagent is membrane permeable. In another embodiment the calcium indicatormolecule is membrane permeable. In another embodiment the source ofcalcium cations is exogenous to the cell. In another embodiment thesample contains at least one compound that uncouples oxidativephosphorylation from ATP production. In another embodiment the cellcomprises at least one polypeptide that is a member of the Bcl-2 family.In another embodiment the cell expresses a gene encoding a polypeptidethat regulates cytosolic calcium. In another embodiment the gene encodesa mitochondrial calcium uniporter. In another embodiment the gene is atransfected gene. In another embodiment the gene encodes a mitochondrialcalcium uniporter. In another embodiment the cell is a permeabilizedcell. In another embodiment the cell adheres to a solid substrate. Inanother embodiment the cell is a non-adherent cell.

In still another embodiment the invention provides a method ofidentifying an agent that uncouples oxidative phosphorylation from ATPproduction, comprising: (a) contacting (i) a biological samplecomprising a cell containing cytosol, a mitochondrion and a calciumindicator molecule, under conditions that permit maintenance ofmitochondrial membrane potential, with (ii) a source of calcium cations,wherein the calcium indicator molecule is capable of generating adetectable signal that is proportional to the level of calcium in thecytosol; (b) detecting the signal generated by the calcium indicatormolecule at a plurality of time points; (c) repeating steps (a) and (b)at least once; and (d) comparing (i) the signal generated by the calciumindicator molecule at one or more of the time points prior to andfollowing at least one of the contacting steps in the absence of thecandidate agent, to (ii) the signal generated by the calcium indicatormolecule at one or more of the time points prior to and following atleast one of the contacting steps in the presence of the candidateagent, wherein an increased level of calcium in the cytosol at a timepoint prior to a contacting step in the presence of the agent, comparedto the level of calcium in the cytosol prior to a contacting step in theabsence of the agent, indicates an agent that uncouples oxidativephosphorylation from ATP production.

In another embodiment the invention provides a method of identifying anagent that is a respiratory inhibitor, comprising: (a)contacting (i) abiological sample comprising a cell containing cytosol, a mitochondrionand a calcium indicator molecule, under conditions that permitmaintenance of mitochondrial membrane potential, with (ii) a source ofcalcium cations, wherein the calcium indicator molecule is capable ofgenerating a detectable signal that is proportional to the level ofcalcium in the cytosol; (b) detecting the signal generated by thecalcium indicator molecule at a plurality of time points; (c) repeatingsteps (a) and (b) at least once; and (d) comparing (i) the signalgenerated by the calcium indicator molecule at one or more of the timepoints prior to and following at least one of the contacting steps inthe absence of the candidate agent, to (ii) the signal generated by thecalcium indicator molecule at one or more of the time points prior toand following at least one of the contacting steps in the presence ofthe candidate agent, wherein an increased level of calcium in thecytosol at a time point prior to a contacting step in the presence ofthe agent, compared to the level of calcium in the cytosol prior to acontacting step in the absence of the agent, indicates an agent that isa respiratory inhibitor.

In another embodiment the invention provides a method of identifying anagent that alters a mitochondrial calcium uniporter, comprising (a)contacting, (i) a biological sample comprising a cell containingcytosol, a mitochondrion and a calcium indicator molecule, underconditions that permit maintenance of mitochondrial membrane potential,with (ii) a source of calcium cations, wherein the calcium indicatormolecule is capable of generating a detectable signal that isproportional to the level of calcium in the cytosol; (b) detecting thesignal generated by the calcium indicator molecule at a plurality oftime points; (c) repeating steps (a) and (b) at least once; and (d)comparing (i) the signal generated by the calcium indicator molecule atone or more of the time points prior to and following at least one ofthe contacting steps in the absence of the candidate agent, to (ii) thesignal generated by the calcium indicator molecule at one or more of thetime points prior to and following at least one of the contacting stepsin the presence of the candidate agent, wherein an increased level ofcalcium in the cytosol at a time point following a contacting step inthe presence of the agent, compared to the level of calcium in thecytosol following a contacting step in the absence of the agent,indicates that the agent alters a mitochondrial calcium uniporter.

In another embodiment the invention provides a method of identifying anagent that alters mitochondrial function, comprising: (a) contacting, ineach of a plurality of reaction vessels in a high throughput screeningarray, (i) a biological sample comprising a cell containing amitochondrion, cytosol and a calcium indicator molecule, underconditions that permit maintenance of mitochondrial membrane potential,and wherein the calcium indicator molecule is membrane permeable andcapable of generating a detectable signal that is proportional to thelevel of calcium in the cytosol, with (ii) a calcium ionophore, underconditions and for a time sufficient to increase calcium levels withinthe cell; (b) detecting in each reaction vessel the signal generated bythe calcium indicator molecule at a plurality of time points; and (c)comparing the signal generated by the calcium indicator molecule at oneor more of the time points in the absence of the candidate agent, to thesignal generated by the calcium indicator molecule at one or more of thetime points in the presence of the candidate agent, and therefromidentifying an agent that alters mitochondrial function.

In another embodiment the calcium ionophore is ionomycin, A23187, NMDAor a cell depolarization signal. In another embodiment the step ofcontacting is repeated at least once. In another embodiment the samplecontains at least one compound that alters intracellular distribution ofa calcium cation. In another embodiment the compound that altersintracellular calcium cation distribution is thapsigargin or Ru360. Inanother embodiment the compound that alters intracellular calcium cationdistribution is a calcium ionophore or a membrane permeable compoundthat alters intracellular calcium distribution. In another embodimentthe sample contains at least one compound that uncouples oxidativephosphorylation from ATP production. In another embodiment the candidateagent is membrane permeable. In another embodiment the source of calciumcations is exogenous to the cell. In another embodiment the samplecontains at least one compound that uncouples oxidative phosphorylationfrom ATP production. In another embodiment the cell comprises at leastone polypeptide that is a member of the Bcl-2 family. In anotherembodiment the cell expresses a gene encoding a polypeptide thatregulates cytosolic calcium. In certain further embodiments the geneencodes a mitochondrial calcium uniporter, and in certain otherembodiments the gene is a transfected gene. In certain furtherembodiments the gene encodes a mitochondrial calcium uniporter. Inanother embodiment the cell adheres to a solid substrate, and in certainother embodiments the cell is a non-adherent cell.

In another embodiment the invention provides a method of identifying anagent that uncouples oxidative phosphorylation from ATP production,comprising (a) contacting (i) a biological sample comprising a cellcontaining cytosol, a mitochondrion and a calcium indicator molecule,under conditions that permit maintenance of mitochondrial membranepotential, and wherein the calcium indicator molecule is membranepermeable and capable of generating a detectable signal that isproportional to the level of calcium in the cytosol, with (ii) a calciumionophore, under conditions and for a time sufficient to increasecalcium levels within the cell; (b) detecting the signal generated bythe calcium indicator molecule at a plurality of time points; (c)repeating steps (a) and (b) at least once; and (d) comparing (i) thesignal generated by the calcium indicator molecule at one or more of thetime points prior to and following at least one of the contacting stepsin the absence of the candidate agent to (ii) the signal generated bythe calcium indicator molecule at one or more of the time points priorto and following at least one of the contacting steps in the presence ofthe candidate agent, wherein an increased level of calcium in thecytosol at a time point prior to a contacting step in the presence ofthe agent, compared to the level of calcium in the cytosol prior to acontacting step in the absence of the agent, indicates an agent thatuncouples oxidative phosphorylation from ATP production.

In another embodiment the invention provides a method of identifying anagent that is a respiratory inhibitor, comprising: (a) contacting (i) abiological sample comprising a cell containing cytosol, a mitochondrionand a calcium indicator molecule, under conditions that permitmaintenance of mitochondrial membrane potential, and wherein the calciumindicator molecule is membrane permeable and capable of generating adetectable signal that is proportional to the level of calcium in thecytosol, with (ii) a calcium ionophore, under conditions and for a timesufficient to increase calcium levels within the cell; (b) detecting thesignal generated by the calcium indicator molecule at a plurality oftime points; (c) repeating steps (a) and (b) at least once; and (d)comparing (i) the signal generated by the calcium indicator molecule atone or more of the time points prior to and following at least one ofthe contacting steps in the absence of the candidate agent to (ii) thesignal generated by the calcium indicator molecule at one or more of thetime points prior to and following at least one of the contacting stepsin the presence of the candidate agent, wherein an increased level ofcalcium in the cytosol at a time point prior to a contacting step in thepresence of the agent, compared to the level of calcium in the cytosolprior to a contacting step in the absence of the agent, indicates anagent that is a respiratory inhibitor.

In another embodiment the invention provides a method of identifying anagent that alters a mitochondrial calcium uniporter, comprising (a)contacting (i) a biological sample comprising a cell containing cytosol,a mitochondrion and a calcium indicator molecule, under conditions thatpermit maintenance of mitochondrial membrane potential, and wherein thecalcium indicator molecule is membrane permeable and capable ofgenerating a detectable signal that is proportional to the level ofcalcium in the cytosol, with (ii) a calcium ionophore, under conditionsand for a time sufficient to increase calcium levels within the cell;(b) detecting the signal generated by the calcium indicator molecule ata plurality of time points; (c) repeating steps (a) and (b) at leastonce; and (d) comparing (i) the signal generated by the calciumindicator molecule at one or more of the time points prior to andfollowing at least one of the contacting steps in the absence of thecandidate agent to (ii) the signal generated by the calcium indicatormolecule at one or more of the time points prior to and following atleast one of the contacting steps in the presence of the candidateagent, wherein an increased level of calcium in the cytosol at a timepoint following a contacting step in the presence of the agent, comparedto the level of calcium in the cytosol following a contacting step inthe absence of the agent, indicates that the agent alters amitochondrial calcium uniporter.

It is another aspect of the invention to provide a method of identifyingan agent that alters mitochondrial function, comprising (a) contacting(i) a biological sample comprising a permeabilized cell depleted ofcytosol, a mitochondrion and a calcium indicator molecule, underconditions that permit maintenance of mitochondrial membrane potential,with (ii) a source of calcium cations, wherein the calcium indicatormolecule is capable of generating a detectable signal that isproportional to the level of calcium in the cell; (b) detecting thesignal generated by the calcium indicator molecule at a plurality oftime points; and (c) comparing the signal generated by the calciumindicator molecule at one or more of the time points in the absence of acandidate agent, to the signal generated by the calcium indicatormolecule at one or more of the time points in the presence of thecandidate agent, and therefrom identifying an agent that altersmitochondrial function. In another related embodiment there is providedsuch a method in a high throughput screening format, wherein the step ofcontacting is performed in each of a plurality of reaction vessels in ahigh throughput screening array, and the step of detecting is performedin each reaction vessel.

According to certain further embodiments, the calcium indicator moleculeis capable of generating a detectable signal that is proportional eitherto the level of calcium in the mitochondrion or to the level of calciumoutside of the mitochondrion. In certain other further embodiments thestep of contacting is repeated at least once. In certain other furtherembodiments the sample contains at least one compound that altersintracellular distribution of a calcium cation. In a still furtherembodiment the compound that alters intracellular calcium cationdistribution is thapsigargin or Ru360. In another embodiment the samplecontains at least one compound that uncouples oxidative phosphorylationfrom ATP production. In another embodiment the source of calcium cationsis exogenous to the cell, and in another embodiment the sample containsat least one compound that uncouples oxidative phosphorylation from ATPproduction. In certain embodiments the cell comprises at least onepolypeptide that is a Bcl-2 family member, and in certain otherembodiments the cell expresses a gene encoding a polypeptide thatregulates cytosolic calcium. In a further embodiment the gene encodes amitochondrial calcium uniporter, and in a distinct further embodimentthe gene is a transfected gene. In a still further embodiment the geneencodes a mitochondrial calcium uniporter. According to certainembodiments the cell adheres to a solid substrate, while in certainother embodiments the cell is a non-adherent cell.

In another embodiment the present invention provides a method ofidentifying an agent that alters mitochondrial function, comprising (a)contacting (i) a biological sample comprising one or more isolatedmitochondria and a calcium indicator molecule in a medium, underconditions that permit maintenance of mitochondrial membrane potential,with (ii) a source of calcium cations, wherein the calcium indicatormolecule is capable of generating a detectable signal that isproportional to the level of calcium in the biological sample; (b)detecting the signal generated by the calcium indicator molecule at aplurality of time points; and (c) comparing the signal generated by thecalcium indicator molecule at one or more of the time points in theabsence of a candidate agent, to the signal generated by the calciumindicator molecule at one or more of the time points in the presence ofthe candidate agent, and therefrom identifying an agent that altersmitochondrial function. In another related embodiment there is providedsuch a method in a high throughput screening format, wherein the step ofcontacting is performed in each of a plurality of reaction vessels in ahigh throughput screening array, and the step of detecting is performedin each reaction vessel.

In certain further embodiments the calcium indicator molecule is capableof generating a detectable signal that is proportional to the level ofcalcium in the mitochondrion, and in certain other further embodimentsthe calcium indicator molecule is capable of generating a detectablesignal that is proportional to the level of calcium outside of themitochondrion. In certain embodiments the step of contacting is repeatedat least once. In another embodiment the sample contains at least onecompound that alters distribution of a calcium cation in the sample,which in certain further embodiments is thapsigargin or Ru360. Inanother embodiment the sample contains at least one compound thatuncouples oxidative phosphorylation from ATP production, and in anotherembodiment the isolated mitochondria are derived from a cell thatcomprises at least one polypeptide that is a Bcl-2 family member. Incertain other embodiments the isolated mitochondria are derived from acell that expresses a gene encoding a polypeptide that regulatescytosolic calcium, and in certain further embodiments the gene encodes amitochondrial calcium uniporter. In certain other embodiments the geneis a transfected gene, and in certain further embodiments the geneencodes a mitochondrial calcium uniporter.

In certain further embodiments of invention methods described above,subsequent to the step of contacting the biological sample with thesource of calcium cations and prior to the step of comparing signals,the biological sample is contacted (i) with at least one compound thatuncouples oxidative phosphorylation from ATP production, and (ii) withat least one agent that alters mitochondrial function. In someembodiments the agent that alters mitochondrial function is cyclosporinA, and in certain other embodiments the agent is cyclosporin A,rotenone, oligomycin, succinate or Bcl-2. In certain embodiments thecompound that uncouples oxidative phosphorylation from ATP production isFCCP or CCCP.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All references disclosed herein are hereby incorporated byreference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the entry and distribution of calcium ions (Ca²⁺,represented as “●” in the figure) into permeabilized cells. Othersymbols and abbreviations: dashed line, permeabilized cell membrane;ovals, mitochondria; rectangle with solid border, extramitochondrial(EM) calcium reservoir; CaUP, mitochondrial calcium uniporter.

FIG. 2 depicts the entry of a reagent (represented as “◯” in the figure)capable of providing a detectable signal in a calcium-sensitive mannerinto permeabilized cells. When a molecule of the calcium-sensitivereagent combines with a calcium ion, detectable signals (asterisks) areemitted. Other symbols and abbreviations, same as in FIG. 1. Calciumions in the media and the cytosolic space are detected in thisembodiment of the assay, but the calcium-sensitive detectable reagentcannot enter mitochondria and other (extramitochondrial) calciumreservoirs, and calcium ions sequestered in mitochondria orextramitochondrial calcium reservoirs are thus not detected.

FIG. 3 depicts an embodiment of the assay of the invention in which anextramitochondrial (EM) calcium reservoir releasing agent is present.Symbols, same as in FIGS. 1 and 2. In this embodiment of the assay,calcium ions sequestered in EM calcium reservoirs are released therefromand/or prevented from entering such reservoirs. As a result,mitochondria are a sole or main site of sequestration of calcium ions,and changes in the levels of detectable calcium ions correspond solelyor at least mainly to the influx (or efflux) of Ca²⁺ into (or from)mitochondria. In the figure, calcium ions are shown enteringmitochondria due to the action of the calcium uniporter (CaUP).

FIG. 4 is a schematic reflecting idealized results of an assay of theinvention. The initial Ca²⁺ pulse is shown in panel A, wherein thesignal corresponding to extramitochondrial Ca²⁺ decreases soon aftercalcium is added as the calcium uniporter (CaUP) acts to sequester Ca²⁺ions in mitochondria. In panel B, the test compounds added to thepermeabilized cells are respiratory uncouplers or inhibitors, whichcauses the CaUP to act in reverse; that is, under these conditions, CaUPstimulates the release of Ca²⁺ from, instead of uptake into,mitochondria. This release of mitochondrial Ca²⁺ causes the signalcorresponding to extramitochondrial Ca²⁺ to increase immediately or soonafter the test compound is added (dotted line indicates results in theabsence of a test compound). In panel C, the test compound has no effecton respiration or the calcium uniporter, as shown by the fact that asecond pulse of Ca²⁺ is taken up by mitochondria with the same kineticprofile as in the initial Ca²⁺ pulse; the same result would occur if nocompound were added before the second pulse of Ca²⁺. In panel D, thetest compounds are, as indicated, either inhibitors or stimulators ofthe calcium uniporter, and the dotted line indicates the resultsexpected in the absence of test compound.

FIG. 5 shows results obtained with one of the assays of the inventionwherein MixCon cells were challenged with increasing concentrations (0to 16 uM) of calcium ions.

FIG. 6 shows results obtained with one of the assays of the inventionwherein MixCon cells were challenged with increasing concentrations (0to 20 uM) of calcium ions.

FIG. 7 shows the effect of two compounds, rotenone and oligomycin, thatare known to impact mitochondrial functions, on the assay of theinvention.

FIG. 8 shows the effects of different optional media components (malate,succinate and glutamate), in various combinations with rotenone andoligomycin.

FIG. 9 shows the results of efforts to optimize the concentrations ofcalcium ions, rotenone and oligomycin for the assay.

FIG. 10 shows the effect of increasing concentrations of FCCP on theassay of the invention.

FIG. 11 shows the data presented in FIG. 10 plotted as a function oftime.

FIG. 12 shows the impact of various agents on calcium levels with(columns 7-12) and without (1-6) thapsigargin.

FIG. 13 shows the impact of an MPT effector, diamide, on resultsobtained using the assay of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for identifying compounds thatalter mitochondrial function, and in particular, that influencemitochondrial regulation of intracellular calcium levels, including highthroughput screening assays for the detection of the physiological orpharmacological effect of a candidate agent. The invention is based inpart on the unexpected adaptability of sensitive, cell-based metrictechniques to a high throughput format, and on the ability to modifythese techniques for screening panels of candidate agents such as drugsand pharmacophores. Additionally, the invention is based in part on thesurprising observation that, through the use of a compound that altersintracellular calcium cation distribution, selective conditions can bedevised for monitoring regulation by mitochondria of intracellularcalcium in cell-based assays such as high throughput drug screening.

According to the present invention, a cell that contains cytosol, amitochondrion and a calcium indicator molecule (or in certainembodiments, a permeabilized cell containing mitochondria, or apermeabilized cell containing mitochondria and depleted of cytosol, or asuspension of isolated mitochondria, in each case containing a calciumindicator molecule) is contacted one or more times with a source ofcalcium cations (Ca²⁺) under conditions that permit maintenance ofmitochondrial membrane potential, and a signal generated by the calciumindicator molecule, which signal is proportional to the level of calciumin the cytosol, is detected at a plurality of time points. Mitochondriathat are capable of maintaining a membrane potential can regulatecytosolic free calcium levels, such that monitoring cytosolic calciumlevels as described herein permits screening for and identification ofagents that alter this and related mitochondrial functions, includingmitochondrial calcium uniporter activity. By comparing the signalgenerated in the presence of the candidate agent to the signal generatedin the absence of the agent, detection of an alteration (e.g., anincrease or decrease) in the signal that accompanies the introduction ofthe agent can signify that the agent may usefully alter mitochondrialfunction.

Thus, as described in greater detail below, the present inventionrelates to compositions and methods for screening compounds that mayalter mitochondrial function, including high throughput screeningassays, by detecting changes in intracellular calcium levels that areregulated by mitochondrial activity. The screening assays of the presentinvention include assays that are performed using intact cells, and alsoinclude assays performed using permeabilized cells. As provided herein,in certain embodiments the present invention provides a method foridentifying an agent that alters mitochondrial function, and in certainother embodiments the invention provides a method for identifying anagent that is a respiratory inhibitor. In certain other embodiments theinvention provides a method for identifying an agent that uncouplesoxidative phosphorylation from ATP production. In certain otherembodiments the invention provides a method for identifying an agentthat inhibits a mitochondrial calcium uniporter. Moreover, as disclosedherein, the subject invention methods permit distinguishing betweencertain types of agents that alter mitochondrial function. For example,based on the teachings herein, by detecting the signal generated over aperiod of time by a calcium indicator molecule as provided herein, aperson having ordinary skill in the art can determine whether an agentinhibits a mitochondrial calcium uniporter or whether, alternatively, anagent may be a respiratory inhibitor or an uncoupler of oxidativephosphorylation from ATP production.

According to the present invention, and as described in greater detailbelow, a biological sample comprising a cell comprising cytosol, amitochondrion and a calcium indicator molecule (or in certainembodiments, a permeabilized cell containing mitochondria, or apermeabilized cell containing mitochondria and depleted of cytosol, orin preferred embodiments a suspension of isolated mitochondria, in eachcase containing a calcium indicator molecule), is contacted with asource of calcium cations under conditions that permit maintenance ofmitochondrial membrane potential, and a detectable signal generated bythe calcium indicator molecule and proportional to the level ofcytosolic calcium is detected at a plurality of time points. The step ofcontacting with a source of calcium cations may be optionally repeatedat least once or a plurality of times, and the signal generated by thecalcium indicator molecule at one or more of the time points in theabsence of a candidate agent is compared to the signal generated by thecalcium indicator molecule at one or more of the time points in thepresence of the candidate agent.

According to non-limiting theory, an initial calcium-contacting stepinduces a transient elevation of detectable cytosolic calcium that,under control conditions, dissipates as calcium is imported into themitochondria (FIG. 1A). If a candidate agent has no effect onmitochondrial function, a subsequent step of contacting with a source ofcalcium would similarly result in a transient rise in cytosolic calciumthat dissipates as mitochondrial uptake of this cytosolic calciumproceeds (FIG. 1C). Depending on the particular biological sample andthe quantity of calcium present, after one, two or morecalcium-contacting steps, mitochondrial capacity for calcium uptake maybe supra-saturated such that collapse of mitochondrial membranepotential and mitochondrial membrane permeability transition (MPT) areinduced, leading to a detectable, spontaneous release of calcium frommitochondria into the cytosol. In preferred embodiments of theinvention, at least two or three calcium-contacting steps would berequired to induce this type of spontaneous calcium release.

If a candidate agent is present that uncouples oxidative phosphorylationfrom ATP production (i.e., a “respiratory uncoupler”), mitochondrialmembrane potential dissipates and mitochondria release calcium back intothe cytosol (e.g., via the calcium uniporter), generating a detectableincrease in the signal produced by the calcium indicator molecule (FIG.1B). If, however, a candidate agent is present that is an inhibitor ofthe calcium uniporter, mitochondrial uptake of cytosolic calcium iscompletely or partially impaired following one or severalcalcium-contacting steps, resulting in higher levels of detectablecytosolic calcium (FIG. 1D). Conversely, if a candidate agent is presentthat stimulates, augments or otherwise enhances calcium uniporteractivity, mitochondrial uptake of calcium from the cytosol following acalcium-contacting step is detected as a decreased signal generated bythe calcium indicator molecule (FIG. 1D). In certain embodiments of theinvention, a compound that alters intracellular distribution of calciumcations may optionally be present, for example thapsigargin, rutheniumred (e.g., Ying et al., Biochem. 30:4949, 1991; Matlib et al., J. Biol.Chem. 273:10223, 1998), Ru360 (e.g., Emerson et al., J. Am. Chem. Soc.115:11799, 1993), Bcl-2 (e.g., Murphy et al., Proc. Nat. Acad. Sci. USA93:9893, 1996; U.S. Pat. No. 5,459,251) or one or more other suitablecompounds. Optionally, additional compounds that may alter mitochondrialfunction may also be present, for example,chloromethyltetramethylrosamine (e.g., Scorrano et al., Proc. Nat. Acad.Sci. USA 274:24567, 1999), cyclosporin A (e.g., Petronilli et al.,Biophys. JI. 76:725, 1999; Murphy et al., Proc. Nat. Acad. Sci. USA93:9893), rotenone, oligomycin or succinate (Murphy et al., 1996).

“Altered mitochondrial function” may refer to any condition or state,including those that accompany a disease associated with alteredmitochondrial function, where any structure or activity that is directlyor indirectly related to a mitochondrial function has been changed in astatistically significant manner relative to a control or standard.Altered mitochondrial function may have its origin in extramitochondrialstructures or events as well as in mitochondrial structures or events,in direct interactions between mitochondrial and extramitochondrialgenes and/or their gene products, or in structural or functional changesthat occur as the result of interactions between intermediates that maybe formed as the result of such interactions, including metabolites,catabolites, substrates, precursors, cofactors and the like.

Additionally, altered mitochondrial function may include alteredrespiratory, metabolic or other biochemical or biophysical activity inone or more cells of a biological sample or a biological source. Asnon-limiting examples, markedly impaired ETC activity may be related toaltered mitochondrial function, as may be generation of increasedreactive oxygen species (ROS) or defective oxidative phosphorylation. Asfurther examples, altered mitochondrial membrane potential, induction ofapoptotic pathways and formation of atypical chemical and biochemicalcrosslinked species within a cell, whether by enzymatic or non-enzymaticmechanisms, may all be regarded as indicative of altered mitochondrialfunction. These and other non-limiting examples of altered mitochondrialfunction are contemplated by the present invention.

Without wishing to be bound by theory, altered mitochondrial functionmay be related to altered intracellular calcium regulation that may, forexample, accompany loss of mitochondrial membrane electrochemicalpotential by intracellular calcium flux, by mechanisms that include freeradical oxidation, defects in transmitochondrial membrane shuttles andtransporters such as the adenine nucleotide transporter or themalate-aspartate shuttle, by defects in ATP biosynthesis, by impairedassociation with porin of hexokinases and/or other enzymes or by otherevents. Altered intracellular calcium regulation and/or collapse ofmitochondrial inner membrane potential may result from direct orindirect effects of mitochondrial genes, gene products or relateddownstream mediator molecules and/or extramitochondrial genes, geneproducts or related downstream mediators, or from other known or unknowncauses. Thus, an “indicator of altered mitochondrial function” may beany detectable parameter that directly relates to a condition, process,pathway, dynamic structure, state or other activity involvingmitochondria and that permits detection of altered mitochondrialfunction in a biological sample from a subject or biological source.According to non-limiting theory, altered mitochondrial functiontherefore may also include altered mitochondrial permeability to calciumor to mitochondrial molecular components involved in apoptosis (e.g.,cytochrome c), or other alterations in mitochondrial respiration.

Certain aspects of the present invention as it relates to monitoring theactivity of mitochondrial molecular components that bind, transport orotherwise regulate intracellular calcium involve the relationshipbetween mitochondrial Δψ and intracellular calcium homeostasis. Theinvention thus relates in part to detecting, in a biological samplecomprising a cell containing cytosol, a mitochondrion and a calciumindicator molecule as provided herein, a signal generated by the calciumindicator molecule. Accordingly, in certain preferred embodimentsaltered mitochondrial function may be manifest as altered mitochondrialregulation of intramitochondrial and extramitochondrial calcium levels,as altered mitochondrial calcium uniporter activity, as alteredmitochondrial membrane potential, as altered coupling of mitochondrialoxidative phosphorylation to mitochondrial ATP production, as alteredmitochondrial respiration, or as an alteration (e.g., a statisticallysignificant change relative to a control) in any other mitochondrialfunction or activity as provided herein and as known to the art.

By way of background, fluctuations in mitochondrial calcium are normaloccurrences that are part of intracellular calcium homeostasis, as alsonoted above. Additionally, mitochondrial calcium levels may reflecttransient low cytosolic calcium concentrations, which, in combinationwith reduced ATP or other conditions associated with mitochondrialpathology can yield mitochondrial permeability transition (MPT, see,e.g., Gunter et al., Biochim. Biophys. Acta 1366:5, 1998; Rottenberg etal., Biochim. Biophys. Acta 1016:87, 1990). Generally, under restingconditions the extramitochondrial (i.e., cytosolic) level of Ca²⁺ isgreater than that present within mitochondria. In the case of certaindiseases or disorders, including diseases associated with alteredmitochondrial function, mitochondrial or cytosolic calcium levels mayvary from the above ranges and may range from, e.g., about 1 nM to about500 nM, more typically from about 10 nM to about 100 μM and usually fromabout 20 nM to about 1 μM, where “about” indicates ±10%.

Because loss of membrane potential causes mitochondria to releasesequestered Ca²⁺ into the cytosol, the Ca²⁺ load on nearby mitochondriais increased, setting up a chain reaction (Darley-Usmar et al., Ann.Med. 23:583, 1991). Independent of the pathological sequelae of PTcollapse, which include increased radical production from uncoupledelectron transfer, the ensuing loss of ATP per se may be lethal toaerobically poised cells (Jurkowitz-Alexander et al., J. Neurochem.59:344, 1992). In addition to a reduced metabolic energy supply, thelack of ATP may exacerbate Δψm collapse.

MPT may also be induced by compounds that bind one or more mitochondrialmolecular components. Such compounds include, but are not limited to,atractyloside and bongkrekic acid. Methods of determining appropriateamounts of such compounds to induce MPT are known in the art (see, e.g.,Beutner et al., Biochim. Biophys. Acta 1368:7, 1998; Obatomi and Bach,Toxicol. Lett. 89:155, 1996; Green and Reed, Science 281:1309, 1998;Kroemer et al., Annu. Rev. Physiol. 60:619, 1998; and references citedtherein).

Under certain conditions, a mitochondrial state which can featurealtered mitochondrial regulation of intracellular calcium (e.g., alteredmitochondrial membrane permeability to calcium) may be induced byexposing a biological sample to compositions referred to as “apoptogens”that induce programmed cell death, or “apoptosis”. A variety ofapoptogens are known to those familiar with the art (see, e.g., Green etal., Science 281:1309, 1998, and references cited therein) and mayinclude by way of illustration and not limitation: tumor necrosisfactor-alpha (TNF-α); Fas ligand; glutamate; N-methyl-D-aspartate(NMDA); interleukin-3 (IL-3); herbimycin A (Mancini et al., J. Cell.Biol. 138:449-469, 1997); paraquat (Costantini et al., Toxicology99:1-2, 1995); ethylene glycols; protein kinase inhibitors, such asstaurosporine, calphostin C, caffeic acid phenethyl ester, chelerythrinechloride, genistein; 1-(5-isoquinolinesulfonyl)-2-methylpiperazine;N-[2-((p-bromocinnamyl)amino)ethyl]-5-5-isoquinolinesulfonamide; KN-93;quercitin; d-erythro-sphingosine derivatives, for example, ceramide; UVirradiation; ionophores such as ionomycin and valinomycin; MAP kinaseinducers such as anisomycin, anandamine; cell cycle blockers such asaphidicolin, colcemid, 5-fluorouracil, homoharringtonine;acetylcholinesterase inhibitors such as berberine; anti-estrogens suchas, tamoxifen; pro-oxidants, such as tert-butyl peroxide, hydrogenperoxide; free radicals such as nitric oxide; inorganic metal ions, suchas cadmium; DNA synthesis inhibitors, including, for example,actinomycin D and also including DNA topoisomerase inhibitors, forexample, etoposide; DNA intercalators such as doxorubicin, bleomycinsulfate, hydroxyurea, methotrexate, mitomycin C, camptothecin,daunorubicin; protein synthesis inhibitors such as cycloheximide,puromycin, rapamycin; agents that affect microtubulin formation orstability, for example, vinblastine, vincristine, colchicine,4-hydroxypbenylretinamide, paclitaxel; Bad protein, Bid protein and Baxprotein (see, e.g., Jurgenmeier et al., Proc. Nat. Acad. Sci. USA95:4997-5002, 1998, and references cited therein); calcium and inorganicphosphate (Kroemer et al., Ann. Rev. Physiol. 60:619, 1998).

As noted above, the invention thus pertains in part to detecting asignal generated by a calcium indicator molecule in a biological sampleas provided herein. The calcium indicator molecule may be endogenous to(e.g., naturally occurring in) the sample or it may be exogenous, whichincludes at least one calcium indicator molecule that does not occurnaturally in the biological sample but that has been loaded,administered, admixed, expressed (including expression as the product ofa genetically engineered nucleic acid construct), targeted, contacted,exposed or otherwise artificially introduced into the sample, as long asthe calcium indicator molecule is capable of generating a detectablesignal that is proportional to the level of calcium in the cytosol ormitochondria. In preferred embodiment the calcium indicator molecule isexogenous and the detectable signal is a fluorescent signal.

It is therefore contemplated by the present invention to provide amethod for assaying a cytosolic calcium level in a biological sample, inpertinent part, by contacting a biological sample comprising a cellcontaining cytosol, a mitochondrion and a calcium indicator molecule asprovided herein, under conditions that permit maintenance ofmitochondrial membrane potential, with a source of calcium cations, anddetecting a signal generated by the calcium indicator molecule at aplurality of time points, for example, to generate a time-course ofdetected signal levels. Where the calcium indicator molecule is afluorescent indicator, the signal generated by the indicator molecule,which signal is proportional to the level of calcium in the cytosol, maybe detected by exposing the sample to light having an appropriatewavelength to excite the indicator, and determining resultantfluorescence with a suitable instrument for detecting a fluorescentlight emission at an appropriate wavelength. Those having ordinary skillin the art can readily determine the manner by which the sample iscontacted with the source of calcium cations based on the teachingsprovided herein, in view of the properties of the sample (including thecalcium indicator molecules selected) and those of the source of calciumions selected. As discussed in greater detail below, the method of thepresent invention may be used to identify an agent that altersmitochondrial function, that uncouples oxidative phosphorylation fromATP production, that is a respiratory inhibitor or that alters amitochondrial calcium uniporter.

Thus, in preferred embodiments the calcium indicator molecule may be alight emission molecule, for example a fluorescent, phosphorescent, orchemiluminescent molecule or the like, which emits a detectable signalin the form of light when excited by excitation light of an appropriatewavelength. “Fluorescence” refers to luminescence (emission of light)that is caused by the absorption of radiation at one wavelength(“excitation”), followed by nearly immediate re-radiation (“emission”),usually at a different wavelength, that ceases almost at once when theincident radiation stops. At a molecular level, fluorescence occurs ascertain compounds, known as fluorophores, are taken from a ground stateto a higher state of excitation by light energy; as the molecules returnto their ground state, they emit light, typically at a differentwavelength. “Phosphorescence,” in contrast, refers to luminescence thatis caused by the absorption of radiation at one wavelength followed by adelayed re-radiation that occurs at a different wavelength and continuesfor a noticeable time after the incident radiation stops.“Chemiluminescence” refers to luminescence resulting from a chemicalreaction, and “bioluminescence” refers to the emission of light fromliving organisms or cells, organelles or extracts derived therefrom.

A variety of calcium indicators are known in the art and are suitablefor generating a detectable intracellular signal, for example, a signalthat is proportional to the level of calcium in the cytosol or in themitochondria, depending on a variety of factors pertaining to assayconfiguration, such as the particular biological sample and assayreagents that are selected. Suitable calcium indicators include but neednot be limited to fluorescent indicators such as fura-2 (McCormack etal., 1989 Biochim. Biophys. Acta 973:420); mag-fura-2; BTC (U.S. Pat.No. 5,501,980); fluo-3, fluo-4, fluo-5F and fluo-5N (U.S. Pat. No.5,049,673); fura-4F, fura-5F, fura-6F, and fura-FF; rhod-2, rhod-5F;Calcium Green™ 5N; benzothiaza-1 and benzothiaza-2; and others, whichare available from Molecular Probes, Inc., Eugene, Oreg. (see also,e.g., Calcium Signaling Protocols—Meths. In Mol. Biol.—Vol. 114),Lambert, D. (ed.), Humana Press, 1999). In certain embodiments whereincalcium can be directly measured, a free calcium ion may itself act as acalcium indicator molecule. Such embodiments are directed to adetectable signal that is proportional to the level of calcium that ispresent, as determined, for example, using a calcium sensitive electrode(commercially available from, e.g., World Precision Instrument, Inc.,Sarasota, Fla.) connected to an appropriate meter (e.g., a pH meter);preferably such direct calcium measurements are made when the biologicalsample comprises a permeabilized cell, a permeabilized cell depleted ofcytosol, or one or more isolated mitochondria in a medium.

Calcium Green™ 5N is a particularly preferred calcium indicator moleculefor use according to the present invention. Depending, however, on theparticular assay conditions to be used, a person having ordinary skillin the art can select a suitable calcium indicator from those describedabove or from other calcium indicators, according to the teachingsherein and based on known properties (e.g., solubility, stability, etc.)of such indicators. For example by way of illustration and notlimitation, whether a cell permeant or cell impermeant indicator isneeded (e.g., whether a sample comprises a permeabilized cell), affinityof the indicator for calcium (e.g., dynamic working range of calciumconcentrations within a sample as provided herein) and/or fluorescencespectral properties such as a calcium-dependent fluorescence excitationshift, may all be factors in the selection of a suitable calciumindicator.

A variety of instruments can be used in methods of the invention toexcite a calcium indicator molecule as provided herein that is afluorescent compound, and to detect the signal generated by the calciumindicator molecule that is proportional to the level of calcium in thecytosol, e.g., to measure the resulting emission therefrom. Selection ofa suitable instrument, light source, filter set, etc. may depend onfactors known to those familiar with the art, such as (i) application ofenergy (i.e., light) at a wavelength that will excite the calciumindicator molecule, preferably at or near the optimum excitationwavelength of the indicator molecule (λmax_((ex))); (ii) detection ofenergy (i.e., light) within the emission spectrum of the acceptorcompound, preferably at or near the optimum emission wavelength of theindicator molecule (λmax_((em))); (iii) the type of samples to beassayed; and (iv) the number and formatting of samples to be assayed ina given program, for example, a high throughput screening format.

With regard to factors (i) and (ii), the spectra of energy being appliedto, and the spectra of energy being emitted by the samples willdetermine, in general, what type of instrument will be used. Forexample, although λ(ex) should not be identical to λ(em), the minimalacceptable amount of difference between these two values will beinfluenced by, among other factors, the instrumentation being used. Thatis, as λ(ex) approaches λ(em), instruments capable of resolvingclosely-spaced wavelengths are required, and an assay wherein thedifference between λ(ex) and λ(em) is less than about 3 to about 5 nmrequires a high resolution instrument. Conversely, an assay wherein thedifference between λ(ex) and λ(em) is greater than about 50 to about 75nm requires an instrument having relatively medium to low resolution.

Thus, with specific regard to factor (ii), the type of energy beingemitted by an excited fluorophore and measured in samples willdetermine, in general, what type of instrument will be used. Afluorometer, for instance, is a device that measures fluorescent energyand should therefore be part of the instrumentation. A fluorometer maybe anything from a relatively simple, manually operated instrument thataccommodates only a few reaction vessels (e.g., sample tubes) at a time,to a somewhat more complex manually operated or robotic instrument thataccommodates a larger number of samples in a format having a pluralityof reaction vessels, such as a 96-well microplate (e.g., an fmax™fluorimetric plate reader, Molecular Devices Corp., Sunnyvale, Calif.;or a Cytofluor™ fluorimetric plate reader, model #2350, Millipore Corp.,Bedford, Mass.), or a complex robotic instrument (e.g., a FLIPR™instrument; see infra) that accommodates a multitude of samples in avariety of formats such as 96-well microplates, 384-well microplates orother high throughput screening formats wherein, for example, detectionof signals from a calcium indicator molecule in a plurality or reactionvessels may be automated.

With regard to factor (iii), the type of samples to be assayed in agiven program, different formats will be appropriate for different typesof samples. For example, 96-well or 384-well microplates may be suitablein instances where the cells of interest adhere to the microplatesubstrate, or to some material applied to the wells of the microplate(e.g., a natural or synthetic coating with which the wells have beentreated, such as collagen, fibronectin, vitronectin, RGD peptide,poly-L-lysine, CelTak™, or the like). Interfering fluorescence derivedfrom certain common plastic multiwell plate materials, however, mayresult in a large artifactual background component at excitationwavelengths below about 400 nm. Accordingly, for measurements involvingnonadherent cells such as suspension cells, or suspensions of adherentcells that have been dislodged from a growth substrate, or suspension ofadherent cells on microcarriers or the like, an instrument capable ofreading fluorescent signals in glass or polymeric tubes or tubing, oranother suitable non-interfering vessel, may be preferred. Regardless ofwhat type of format is used, assay reaction vessels should allow for theintroduction of biological samples, candidate agents, a source ofcalcium cations, control reagents and optionally additional compoundsthat may influence cytosolic calcium levels, as well as the ability todetect the signal generated by the calcium indicator molecule at aplurality of appropriate points in time.

Factor (iv), the number of samples to be assayed in a given program, mayinfluence the degree of automation that can be implemented by theinstrument selected. For example, when high throughput (HTS) screening,(i.e., assaying a large number of samples in a relatively brief timeperiod) is desired, robotic or semi-robotic instruments are preferred.Alternatively, samples may be processed manually, even where formatsthat accommodate large sample numbers (e.g., 96-well microplates) areused.

As noted above, the present invention provides assays for use inidentifying agents that alter mitochondrial regulation of intracellularcalcium. Such assays are designed to detect an agent that altersmitochondrial function, a calcium uniporter inhibitor, a respiratoryinhibitor and/or an uncoupler of oxidative phosphorylation from ATPproduction. The invention thus provides efficient methods of identifyingagents, compounds or lead compounds for agents active at the level of amitochondrial calcium regulatory function. Generally, these screeningmethods involve assaying for compounds which alter intracellularcytosolic free calcium levels under conditions that facilitatedetermination of mitochondrial involvement in regulating cytosoliccalcium and, in preferred embodiments, such methods may be directed todetermination of the involvement of a mitochondrial calcium uniporter.The methods are amenable to automated, cost-effective high throughputscreening of chemical libraries for lead compounds.

The term “screening” refers to the use of the invention to identifyagents, for instance, from among large collections of candidate agents,that alter mitochondrial regulation of intracellular calcium in anegative or positive fashion. Briefly, cells or portions thereof thatcomprise cytosol, one or more mitochondria and a calcium indicatormolecule as provided herein are treated with a candidate agent underconditions that permit detection of intracellular calcium levels,including the use of pharmacologic inhibitors (or potentiators) or otherassay reaction components having potentially relevant biologicalactivities, to determine uptake or release of intracellular calcium bymitochondria. The effect of the candidate agent on detectableintracellular calcium levels is then monitored and compared to a controlsample that has been treated identically except for omission of thecandidate agent (e.g., with only the vehicle used to deliver the agent).Detection employs a calcium-sensitive reporter molecule (e.g., a calciumindicator molecule as provided herein) capable of generating adetectable signal that corresponds to the local calcium concentration.

It is contemplated that the present invention will be of major value inhigh throughput screening; i.e., in automated screening of a largenumber of candidate compounds for activity against one or more celltypes. It has particular value, for example, in screening synthetic ornatural product libraries for active compounds. The methods of thepresent invention are therefore amenable to automated, cost-effectivehigh throughput drug screening and have immediate application in a broadrange of pharmaceutical drug development programs. In a preferredembodiment of the invention, the compounds to be screened are organizedin a high throughput screening format such as a 96-well plate format, orother regular two dimensional array, such as a 384-well, 48-well or24-well plate format or an array of test tubes. For high throughputscreening the format is therefore preferably amenable to automation. Itis preferred, for example, that an automated apparatus for use accordingto high throughput screening embodiments of the present invention isunder the control of a computer or other programmable controller. Thecontroller can continuously monitor the results of each step of theprocess, and can automatically alter the testing paradigm in response tothose results.

Depending on the assay, a Fluorometric Imaging Plate Reader (FLIPR™)instrument (Molecular Devices, Sunnyvale, Calif.) is often theinstrument of choice for fluorescence-based assays of the invention. TheFLIPR™ system (see http://www.moleculardevices.com/pages/flipr.html) hasthe following desirable features: (i) It uses a combination of awater-cooled, argon-ion laser illumination and cooled CCD camera as anintegrating detector that accumulates detectable signal over the periodof time in which it is exposed to the image and, as a result, itssignal-to-noise characteristics are generally superior to those ofconventional imaging optics; (ii) it also makes use of a proprietarycell-layer isolation optics that allow signal discrimination on a cellmonolayer, thus reducing undesirable extracellular backgroundfluorescence; (iii) it provides data in real-time, and can also providekinetic data (i.e., readings at a plurality of timepoints); (iv) it hasthe ability to simultaneously excite fluorophores in, and read emissionsfrom, all 96 wells of a 96-well microplate; (v) it provides for precisecontrol of temperature and humidity of samples during analysis; (vi) itincludes an integrated state-of-the-art 96-well pipettor, which usesdispensable tips to eliminate carryover between experiments, and thatcan be used to aspirate, dispense and mix precise volumes of fluids frommicroplates; and, (vii) in the case of the FLIPR³⁸⁴ instrument, it canbe adapted to run sample assays in a robotic or semi-robotic fashion,thus providing for rapid HTS analysis of large numbers of samples (e.g.,up to about a hundred 96-well microplates per day).

As also described above, Ca²⁺ influx into mitochondria appears to belargely dependent, and may be completely dependent, upon the negativetransmembrane electrochemical potential (Δψ) established by electrontransfer, and such influx fails to occur in the absence of Δψ even whenan eight-fold Ca²⁺ concentration gradient is imposed (Kapus et al., FEBSLett. 282:61, 1991). In preferred embodiments of the invention,therefore, a biological sample as provided herein is contacted with asource of calcium cations under conditions that permit maintenance ofmitochondrial membrane potential, as described in greater detail below.Accordingly, mitochondria may release Ca²⁺ via the calcium uniporterdescribed herein when the membrane potential is dissipated, as occurs,for example, with uncouplers of oxidative phosphorylation (i.e.,uncouplers of the mitochondrial ETC from ATP production by ADPphosphorylation) such as 2,4-dinitrophenol (DNP) and carbonyl cyanidep-trifluoro-methoxyphenylhydrazone (FCCP).

A compound that may be a source of calcium cations, according to certainembodiments of the invention, induces increased intracellular,cytoplasmic, cytosolic and/or mitochondrial concentrations of Ca²⁺ byeffecting a redistribution of calcium that is present in theextracellular milieu and/or that is present in one or more of thevarious intracellular compartments. Such compounds, including calciumionophores, are well known to those having ordinary skill in the art.Also provided herein and known to the art are methods for measuringintracellular calcium (see, e.g., Gunter et al., J. Bioenerg Biomembr.26:471, 1994; Gunter et al., Biochim. Biophys. Acta 1366:5, 1998;McCormack et al., Biochim. Biophys. Acta 973:420, 1989; Orrenius et al.,J. Neural. Transm. Suppl. 43:1, 1994; Leist et al., Rev. Physiol.Biochem. Pharmacol. 132:79, 1998; and Haugland, 1996, supra). Examplesof useful calcium ionophores include A23187, ionomycin, CA 1001,enniatin B from Fusarium orthoceras var. enniatum (e.g., Levy et al.,Biochem. Pharmacol. 50:2105, 1995), palytoxin from Palythoa toxica(e.g., Aizu et al., Japan. Jl. Pharmacol. 60:9, 1992), and inappropriate cell types, N-methyl-D-aspartic acid (NMDA) or other celldepolarization signals as known in the art (e.g., Brini et al., NatureMedicine 5:951, 1999) and described herein.

Accordingly, a person skilled in the art may readily select anappropriate procedure for detecting intracellular calcium and a suitableionophore for use as a source of calcium cations in certain embodimentsof the present invention, according to the instant disclosure and towell known methods, including the use of suitable calcium-containingbuffers, media and similar reagents. In addition to ionophores, othercompounds that induce increased intracellular (e.g., cytosolic)concentrations of Ca²⁺ include but are not limited to the sesquiterpenelactone, thapsigargin, which is believed to inhibit sequestration ofcytosolic free calcium in the endoplasmic reticulum (ER), possibly byinhibiting endoplasmic reticular Ca²⁺-ATPase, without blocking calciumrelease by the ER into the cytosol (see, e.g., Takemura et al., J. Biol.Chem. 264:12266, 1989; Thastrup et al., Agents Actions 27:17, 1989; Wonet al., Endocrinol. 136:5399, 1995; Begum et al., J. Biol. Chem.268:3552, 1995; Low et al., Eur. J. Pharmacol. 250:53, 1993). Additionalcompounds capable of increasing or effecting the redistribution ofintracellular calcium include carbachol (e.g., Jence et al., J.Neurochem. 64:1605, 1995; Yan et al., Mol. Pharmacol. 47:248, 1995), BHQ(2,5-Di-(t-butyl)-1,4-hydroquinone; e.g., Salvador et al., Arch.Biochem. Biophys. 351:272, 1998), CPA (cyclopiazonic acid, e.g., Badaouiet al., J. Mol. Cell. Cardiol. 27:2495, 1995) and, in the case of cellshaving appropriate receptors, amino acid neurotransmitters such asglutamate or NMDA.

As will therefore be appreciated by those familiar with the art, theparticular cells that are exposed to a given compound (e.g., glutamate)require a specific receptor therefor (e.g., glutamate receptor), inorder for the compound to influence intracellular Ca²⁺ levels. Forexample, NT-2 teratocarcinoma cells express glutamate receptors, whereasSH-SY5Y neuroblastoma cells do not. Thus, the choice of cell line inwhich it may be desirable to increase intracellular calcium levels willdetermine which compounds are most appropriate.

For example, by way of illustration and not limitation, in certainpreferred embodiments of the invention related to determination ofmitochondrial. regulation of intracellular calcium, ionomycin (Toeplitzet al., J. Amer. Chem. Soc. 101:3344, 1979) may be used as a calciumionophore that provides a source of calcium cations and Fura-2 or Rhod-2(Haugland, 1996 Handbook of Fluorescent Probes and ResearchChemicals-Sixth Ed., Molecular Probes, Eugene, Oreg., pp. 266-274) maybe a fluorescent calcium indicator molecule for detecting cytosolic orintramitochondrial calcium, respectively. In general, any combination ofat least one suitable compound that provides a source of calcium cations(i.e., resulting in increased intracellular concentrations of Ca²⁺) andat least one calcium indicator molecule (i.e., for detectingintracellular calcium levels) that permits measuring mitochondrialregulation of calcium homeostasis in a biological sample may be used. Itis known in the art how to determine suitable concentrations of suchcompounds for the uses contemplated herein (see, e.g., Takei et al.,Brain Res. 652:65, 1994; Hatanaka et al., Biochem. Biophys. Res. Commun.227:513, 1996).

Additionally, pharmacologically active compounds that alter (e.g.,increase or decrease) mitochondrial functions such as ETC activity(e.g., rotenone, oligomycin), or that alter intracellular distributionof Ca²⁺ (e.g., thapsigargin), and with which those skilled in the artwill be familiar, may be optionally employed to assess their effects onmitochondrial regulation of cytosolic calcium. According to non-limitingtheory, such pharmacologically agents may be employed to functionallyisolate calcium pools that are regulated by mitochondria, therebypermitting detection of a relationship between mitochondrial functionand cytosolic calcium levels. For example, a suitable concentration ofthapsigargin may be selected as disclosed herein and known in the art,such that calcium uptake by the endoplasmic reticulum is inhibited,thereby providing detection via the calcium indicator molecule ofmitochondrial calcium loading from extramitochondrial (e.g., cytosolic)pools and/or mitochondrial release of calcium into the cytosol. Numerousvariations in these and related methods and compositions, within thescope of the appended claims, will occur to those skilled in the art, inlight of the present disclosure.

As used herein, mitochondria are comprised of “mitochondrial molecularcomponents”, which may be a protein, polypeptide, peptide, amino acid,or derivative thereof; a lipid, fatty acid or the like, or derivativethereof; a carbohydrate, saccharide or the like or derivative thereof, anucleic acid, nucleotide, nucleoside, purine, pyrimidine or relatedmolecule, or derivative thereof, or the like; or another biologicalmolecule that is a constituent of a mitochondrion. “Mitochondrialmolecular components” includes but is not limited to “mitochondrial porecomponents”. A “mitochondrial pore component” is any mitochondrialmolecular component that regulates the selective permeabilitycharacteristic of mitochondrial membranes as described above, includingthose that bind calcium, transport calcium or are otherwise involved inthe maintenance of calcium and/or other ion levels on either side of themitochondrial membrane. Mitochondrial pore components also includemitochondrial molecular components responsible for establishing Δψm andthose that are functionally altered during MPT.

Isolation and, optionally, identification and/or characterization of themitochondrial pore component or components with which an agent thataffects mitochondrial pore activity interacts may also be desirable andare within the scope of the invention. Once an agent is shown to alter amitochondrial activity such as mitochondrial permeability properties,for example, mitochondrial binding, transport or regulation of calciumas provided herein and in U.S. application Ser. Nos. 09/161,172,09/338,122 and 09/434,3564 or, for example, MPT according to the methodsprovided herein and in U.S. application Ser. No. 09/161,172, thosehaving ordinary skill in the art will be familiar with a variety ofapproaches that may be routinely employed to isolate the molecularspecies specifically recognized by such an agent and involved inregulation of MPT, where to “isolate” as used herein refers toseparation of such molecular species from the natural biologicalenvironment.

Techniques for isolating a mitochondrial molecular component may includeany biological and/or biochemical methods useful for separating thecomponent from its biological source, and subsequent characterizationmay be performed according to standard biochemical and molecular biologyprocedures. Those familiar with the art will be able to select anappropriate method depending on the biological starting material andother factors. Such methods may include, but need not be limited to,radiolabeling or otherwise detectably labeling cellular andmitochondrial components in a biological sample, cell fractionation,density sedimentation, differential extraction, salt precipitation,ultrafiltration, gel filtration, ion-exchange chromatography, partitionchromatography, hydrophobic chromatography, electrophoresis, affinitytechniques or any other suitable separation method that can be adaptedfor use with the agent with which the mitochondrial pore componentinteracts. Antibodies to partially purified components may be developedaccording to methods known in the art and may be used to detect and/orto isolate such components.

A “biological sample comprising a cell containing cytosol, amitochondrion and a calcium indicator molecule” may comprise any tissueor cell preparation in which cells are present that contain (i) cytosol,i.e., any or all intracellular but extraorganellar (e.g.,extramitochondrial, extranuclear, etc.) material, which may includepreferably intracellular sols, cell sap or other solutions, and whichmay also include exogenously derived materials that have been introducedinto an intracellular but extraorganellar localization, for example, inthe case of permeabilized cells, material that by virtue of thepermeabilized state of the cells may come to occupy an intracellularsite; (ii) a calcium indicator molecule as described herein, and (iii)intact mitochondria capable of maintaining a membrane potential whensupplied with one or more oxidizable substrates, for example, glucose,malate, glutamate, pyruvate or galactose.

Mitochondrial membrane potential may be determined according to methodswith which those skilled in the art will be readily familiar, includingbut not limited to detection and/or measurement of detectable compoundssuch as fluorescent indicators, optical probes and/or sensitive pH andion-selective electrodes. (See, e.g., Bernardi et al., Eur. J. Biochem.264:687, 1999; Bernardi, Physiol. Rev. 79:1127, 1999; Ernster et al., J.Cell Biol. 91:227s, 1981, and references cited therein; see alsoHaugland, 1996 Handbook of Fluorescent Probes and ResearchChemicals-Sixth Ed., Molecular Probes, Eugene, Oreg., pp. 266-274 and589-594.) By “capable of maintaining a potential” it is meant that suchmitochondria have a membrane potential that is sufficient to permit theaccumulation of a detectable, potential-sensitive or potentiometriccompound, for example, the fluorescent dyes rhodamine 123, DASPMI[2-,4-dimethylaminostyryl-N-methylpyridinium], TMRM [tetramethylrhodamine methyl ester] or other suitable compounds (see, e.g.,Scheffler, Mitochondria, 1999 Wiley-Liss, NY, pp. 198-202; see alsoHaugland, 1996).

A biological sample comprising a cell containing cytosol and amitochondrion may be derived from a subject or biological source asprovided herein, and subsequently contacted with a calcium indicatormolecule as described herein to provide a biological sample comprising acell containing cytosol, a mitochondrion and a calcium indicatormolecule. As described in greater detail below, the cell may in certainembodiments be a permeabilized cell, and in certain other embodimentsmay be a permeabilized cell depleted of cytosol.

According to certain other embodiments, and as described in greaterdetail below, a “biological sample comprising one or more isolatedmitochondria and a calcium indicator molecule in a medium” (e.g., arespiratory medium) may be a liquid suspension containing mitochondriathat are derived from a subject or biological source as provided herein.In preferred embodiments the isolated mitochondria may be prepared andsubsequently contacted with a calcium indicator molecule to provide abiological sample comprising at least one isolated mitochondrion and acalcium indicator molecule in a medium or inside the mitochondrion,which in preferred embodiments refers to a liquid medium and mayinclude, for example, any of a wide variety of aqueous biologicalbuffers or liquid culture media. In certain other embodiments thecalcium indicator molecule may be present in the isolated mitochondriaat the time of isolation (e.g., recombinantly expressed, mitochondriallytargeted aequorin). In either instance, the biological sample comprisingone or more isolated mitochondria is preferably provided as a liquidsuspension, according to these and related embodiments, such thatintramitochondrial and/or extramitochondrial levels of calcium in thesample may be determined.

Thus, for example, a biological sample may be derived from a normal(i.e., healthy) individual or from an individual having a diseaseassociated with altered mitochondrial function. Biological samples maybe derived by obtaining a blood sample, biopsy specimen, tissue explant,organ culture or any other tissue or cell preparation from a subject ora biological source. The subject or biological source may be abiological organism such as a human or non-human animal, a prokaryote ora eukaryote, a plant, a unicellular organism or a multicellularorganism. According to certain embodiments, the invention contemplates abiological sample comprising in pertinent part a calcium indicatormolecule that is a polypeptide, cofactor, metabolite or the like whichis present in the sample as a biosynthetic product, either naturally oras the result of genetic engineering, such that a suitable biologicalsample may be derived from a biological source without the need for asubsequent step of being contacted with an independently derived calciumindicator molecule.

The subject or biological source may also be a primary cell culture orculture adapted cell line including but not limited to geneticallyengineered cell lines that may contain chromosomally integrated orepisomal recombinant nucleic acid sequences (including but not limitedto a nucleic acid sequence encoding a polypeptide that may be a calciumindicator molecule as provided herein, for example, a green fluorescentprotein (GFP), a FLASH protein or an aequorin-derived polypeptide orfusion protein as provided, for example, in U.S. application Ser. No.09/434,354 and references cited therein), immortalized or immortalizablecell lines, somatic cell hybrid or cytoplasmic hybrid “cybrid” celllines (e.g., U.S. Pat. No. 5,888,498), differentiated ordifferentiatable cell lines, transformed cell lines and the like.

In certain embodiments, for example, a biological sample cell may betransfected with a gene encoding and expressing a biological receptor ofinterest, which may be a receptor having a known ligand (e.g., acytokine, hormone or growth factor) or which may be an “orphaned”receptor for which no ligand is known. Further to such embodiments, oneor more known ligands or other compounds suspected of being able tointeract with the receptor of interest may be optionally included in thesubject invention method, for example, a cytokine, hormone, growthfactor, antibody, neurotransmitter, receptor activator, receptorinhibitor, ion channel modulator, ion pump modulator, irritant, drug,toxin or any other compound known to have, or suspected of having, abiologically relevant activity.

In certain other embodiments, a biological sample cell may express, maybe induced to express or may be transfected with a gene encoding andexpressing a calcium regulatory protein. Calcium regulatory proteinsinclude any naturally occurring or artificially engineered polypeptideor protein that directly or indirectly alter (e.g., increase ordecrease) intracellular or intraorganellar calcium levels. Examples ofcalcium regulatory proteins include calmodulin, calsequestrin, calpainsI and II, calpastatin, calbindin-D_(9k), osteocalcin, osteonectin, S-100protein, troponin C and numerous transmembrane calcium channels. Calciumregulatory proteins also include the mitochondrial calcium uniporter andthe mitochondrial sodium-dependent and sodium-independent calciumtransporters that mediate calcium efflux from mitochondria. Calciumuniporter function may play a role in a variety of normal metabolicprocesses, in apoptosis and in certain disease mechanisms. Although themitochondrial calcium uniporter calcium transport activity has thus beencharacterized, including its activation by ADP, inhibition by ATP, Mg²⁺,ruthenium red and its derivative Ru360 (Matlib et al., J. Biol. Chem.273:10223, 1998; Emerson et al., J. Amer. Chem. Soc. 115:11799, 1993)and competitive inhibition by Sr²⁺, Mn²⁺ and La³⁺, no specificpolypeptide has been identified and confirmed as an authenticmitochondrial calcium uniporter, nor has a gene encoding such auniporter been determined. A candidate uniporter polypeptide that, interalia, localizes to mitochondria and is capable of calcium binding,calcium transport and/or other regulation of or by calcium is disclosedin U.S. application Ser. No. 09/427,867.

For example, some transmembrane calcium channels contain functionalpolypeptide domains related to intracellular binding, transport orregulation of free calcium, for instance, calcium-binding, EFHAND, iontransport, ligand channel and/or calmodulin-binding IQ-domains. EFHAND,Ion Channel, Ligand Channel and IQ. For information on ion transport,see, e.g.: Williams et al., Science 257:3898-395, 1992; Jan et al., Cell69:715-718, 1992. For information on calcium binding/transport, see,e.g.: RyRs (ryanodine receptors) Chen et al., J. Biol. Chem.273:14675-14678, 1998. For information on L-type Ca²⁺ channels, see,e.g.: Hockerman et al., Annu. Rev. Pharmcol. Toxicol. 37:361-396, 1997.For information on ligand channels, see, e.g.: Tong, Science267:1510-1512, 1995; regarding IQ, see, e.g., Xie et al., Nature368:306-312, 1994. For information on EFHAND, see, e.g., Persechini etal., Trends Neurosci. 12:462, 1989; Ikura, Trends Biochem. Sci. 21:14,1996; Guerini, Biochem. Biophys. Res. Commun. 235:271; Kakalis et al.,FEBS Lett. 362:55, 1995. Thus, these or other calcium regulatoryproteins may be expressed in a cell present in a biological sample asprovided herein.

Accordingly, cells for use according to the present invention may beprovided as freshly prepared cells derived from a subject or biologicalsource or as cultured cells, and in certain preferred embodiments thecells are cultured cells. As provided herein and known in the art,cultured cells may be adherent cells that naturally adhere to a solidsubstrate, or may be non-adherent cells that may further be maintainedas cells in a suspension of freely growing cells by cultivation in anappropriate cell culture system. In certain preferred embodiments of thepresent invention, the biological sample comprises a cell that is asuspension cell. In other preferred embodiments, populations ofnaturally adherent cells, which may require attachment to a solidsubstrate for growth, are expanded as adherent cells in suitable cultureflasks and subsequently detached from the flask wall with an appropriatedetaching reagent, for use in the assays described herein. In anotherpreferred embodiment of the invention, the naturally adherent cells aregrown on suspension microcarriers, for example, microspherical beads towhich the cells adhere during the growth, or another appropriate cellcultivating system that permits maintenance and/or assay of adherentcells in a suspension. Microcarriers and other products for handlingadherent cells as cell suspensions are known to those familiar with theart and are commercially available from a variety of sources.

According to certain embodiments contemplated by the present invention,a cell may be a permeabilized cell, which includes a cell that has beentreated in a manner that results in loss of plasma membrane selectivepermeability. For example, it may be desirable to permeabilize a cell ina manner that permits calcium cations in the extracellular milieu todiffuse into the cell, as an alternative to the use of a calciumionophore. As another example, certain calcium indicator molecules asprovided herein may not be readily permeable through the plasmamembrane, such that they may efficiently gain entry to the cytosol onlyfollowing permeabilization of the cell. As yet another example, certaincandidate agents being tested according to the method of the presentinvention may not be able to pass through the plasma membrane, such thata permeabilized cell provides a suitable test cell for the potentialeffects of such agent. Those having ordinary skill in the art arefamiliar with methods for permeabilizing cells, for example by way ofillustration and not limitation, through the use of surfactants,detergents, phospholipids, phospholipid binding proteins, enzymes, viralmembrane fusion proteins and the like; through the use of osmoticallyactive agents; by using chemical crosslinking agents; by physicochemicalmethods including electroporation and the like, or by otherpermeabilizing methodologies.

Thus, for instance, cells may be permeabilized using any of a variety ofknown techniques, such as exposure to one or more detergents (e.g.,digitonin, Triton X-100™, NP-40™ octyl glucoside and the like) atconcentrations below those used to lyse cells and solubilize membranes(i.e., below the critical micelle concentration). Certain commontransfection reagents, such as DOTAP, may also be used. ATP can also beused to permeabilize intact cells, as may be low concentrations ofchemicals commonly used as fixatives (e.g., formaldehyde). Accordingly,in certain embodiments of the invention, it may be preferred to useintact cells and in certain other embodiments the use of permeabilizedcells may be preferred.

According to certain embodiments of the present invention there areprovided methods for identifying an agent that alters mitochondrialfunction, for identifying an agent that uncouples oxidativephosphorylation from ATP production, for identifying a respiratoryinhibitor and for identifying a compound that alters a mitochondrialcalcium uniporter, each of said methods comprising in pertinent part theuse of a sample comprising a mitochondrion contained within a cell thatis permeabilized and that is depleted of cytosol. Determination of whena cell is depleted of cytosol may be accomplished by any of a variety ofmethods well known in the art, for example, those described in Fiskum etal. (1980 Proc. Nat. Acad. Sci. USA 77:3430-3434), includingquantitative methods for monitoring the degree of cytosolic depletion bydetermining any of a number of known cytosolic markers, for example, theenzyme lactate dehydrogenase (LDH), or by monitoring the effects of thedepletion method on cellular architecture. Preferably, a cell depletedof cytosol is essentially completely depleted of cytosol, which refersto depletion of cytosol that results in there being no remainingdetectable cytosolic marker associated with the cell, according tocriteria such as those described Fiskum et al. (1980). In otherembodiments, a cell that is depleted of cytosol may be substantiallydepleted of cytosol, which may include a cell from which greater than 40percent, preferably greater than 60 percent, and more preferably greaterthan 75 percent of at least one detectable cytosolic marker (e.g., LDH)is no longer associated with the cell using criteria known to the art,relative to control cells from which cytosol has not been depleted.

Accordingly, it will be appreciated that the invention also contemplatescompositions and methods for detecting agents that alter (e.g., increaseor decrease in a statistically significant manner) mitochondrialfunction, that alter a mitochondrial calcium uniporter, that uncoupleoxidative phosphorylation from ATP production or that inhibitrespiration, and for detecting compounds that alter the activity of suchagents, which methods may relate to reintroducing to a sample comprisinga mitochondrion (e.g., a cytosol depleted cell as provided herein, or anisolated mitochondrion) one or more cytosolic molecular components.Without wishing to be bound by theory, differences in the resultsobtained when cytosol is present and when cytosol has been depleted asobserved in assays for mitochondrial function as described herein, maybe attributable to the presence or activity of one or more cytosolicmolecular components. Such cytosolic components may include, forexample, ATP or other biolochemical molecules such as metabolites,catabolites, intermediates, cofactors, substrates, catalysts and thelike. Such cytosolic components may also include, for example, one ormore of a protein, peptide, glycopeptide or glycoprotein, nucleic acidor polynucleotide (including, for example, DNA or RNA), lipid includinga glycolipid, proteolipid or phospholipid, or a carbohydrate, or anycombination of such species, that may be present in cytosol. Isolationof cytosolic molecular components may be achieved according to any of anumber of well known biochemical and chemical separation strategiesknown to the art, including but not limited to radiolabeling orotherwise detectably tagging cytosolic components in a biologicalsample, or to cell fractionation, density sedimentation, differentialextraction, salt precipitation, ultrafiltration, gel filtration,ion-exchange chromatography, partition chromatography, hydrophobicchromatography, electrophoresis, affinity techniques or any othersuitable separation method. Antibodies to partially purified componentsmay be developed according to methods known in the art and may be usedto detect and/or to isolate such components.

Affinity techniques may be particularly useful in the context of thepresent invention, and may include any method that exploits a specificbinding interaction between a cytosolic component and an agentidentified according to the invention that interacts with the cytosoliccomponent. For example, because agents that influence mitochondrialfunction (or that uncouple oxidative phosphorylation from ATPproduction, or that alter a mitochondrial calcium uniporter, or thatinhibit respiration) can be immobilized on solid phase matrices, anaffinity binding technique for isolation of the cytosolic component(s)may be particularly useful. Alternatively, affinity labeling methods forbiological molecules, in which such a mitochondrial functionally-activeagent may be modified with a reactive moiety, are well known and can bereadily adapted to the interaction between the agent and a cytosoliccomponent, for purposes of introducing into the cytosolic component adetectable and/or recoverable labeling moiety. (See, e.g., PierceCatalog and Handbook, 1994 Pierce Chemical Company, Rockford, Ill.;Scopes, R. K., Protein Purification: Principles and Practice, 1987,Springer-Verlag, New York; and Hermanson, G. T. et al., ImmobilizedAffinity Ligand Techniques, 1992, Academic Press, Inc., California; fordetails regarding techniques for isolating and characterizing biologicalmolecules, including affinity techniques.

Characterization of cytosolic component molecular species, isolated byaffinity techniques described above or by other biochemical methods, maybe accomplished using physicochemical properties of the cytosoliccomponent such as spectrometric absorbance, molecular size and/orcharge, solubility, peptide mapping, sequence analysis and the like.(See, e.g., Scopes, supra.) Additional separation steps for biomoleculesmay be optionally employed to further separate and identify molecularspecies that co-purify with such cytosolic components that influencemitochdondrial or related functions such as those described herein.These are well known in the art and may include any separationmethodology for the isolation of proteins, lipids, nucleic acids,carbohydrates, or other biological molecules of interest, typicallybased on physicochemical properties of the newly identified componentsof the complex. Examples of such methods include RP-HPLC, ion exchangechromatography, hydrophobic interaction chromatography, hydroxyapatitechromatography, native and/or denaturing one- and two-dimensionalelectrophoresis, ultrafiltration, capillary electrophoresis, substrateaffinity chromatography, immunoaffinity chromatography, partitionchromatography or any other useful separation method.

For example, sufficient amounts of a cytosolic protein may be obtainedfor partial structural characterization by microsequencing. Using thesequence data so generated, any of a variety of well known suitablestrategies for further characterizing the cytosolic components may beemployed. For example, nucleic acid probes may be synthesized forscreening one or more appropriately chosen cDNA libraries to detect,isolate and characterize a cDNA encoding such component(s). Otherexamples may include use of the partial sequence data in additionalscreening contexts that are well known in the art for obtainingadditional amino acid and/or nucleotide sequence information. See, e.g.,Molecular Cloning: A Laboratory Manual, Third Edition, edited bySambrook, Fritsch & Maniatis, Cold Spring Harbor Laboratory, 1989. Suchapproaches may further include nucleic acid library screening based onexpression of library sequences as polypeptides, such as binding of suchpolypeptides to mitochondria-active agents identified according to thepresent invention; or phage display screening approaches or dihybridscreening systems based on protein-protein interactions with knownmitochondrial proteins, and the like, any of which may be adapted toscreening for mitochondrially active cytosolic components provided bythe present invention, using routine methodologies with which thosehaving ordinary skill in the art will be familiar. (See, e.g., Bartel etal., In Cellular Interactions in Development: A Practical Approach, Ed.D. A. Harley, 1993 Oxford University Press, Oxford, United Kingdom, pp.153-179, and references cited, therein.) Preferably extracts of culturedcells, and in particularly preferred embodiments extracts of biologicaltissues or organs may be sources of novel mitochondrially activecytosolic proteins or other cytosolic factors. Preferred sources mayinclude blood, brain, fibroblasts, myoblasts, liver cells or other celltypes.

Briefly, for example, and based essentially on Fiskum et al. (1980 Proc.Nat. Acad. Sci. USA 77:3430-3434), for permeabilized assay cells inwhich cytosol is to be retained, aliquots of cell suspensions containingapproximately 30×10⁶ cells may be pelleted by centrifugation at 16,000×gfor two minutes at room temperature, resuspended in basal incubationmedium (125 mM KCl, 20 mM HEPES-KOH, pH7.0, 2 mM K₂HPO₄) and pelletedagain. The intact cell pellet is transferred into a suitable reactionvessel and assayed at 20° C. in a respiratory medium such as a basalincubation medium containing either (i) 5 mM glutamate and 5 mM malate,or (ii) 5 mM succinate and 2 μM rotenone, as respiratory substrates.

For assay cells in which cytosol is to be depleted, cells are pelleted,resuspended and pelleted again as described above, then resuspended inbasal incubation medium containing respiratory substrates and digitonin(e.g., 150 mM sucrose, 50 mM KCl, 20 mM Hepes, 2 mM K₂HPO₄, pH 7.0containing 5 mM glutamate, 5 mM malate, 0.03% digitonin, 4 mM MgCl₂, 1mM EGTA, 3.0 mM ATP). The cell suspension is incubated for 20 minutes atroom temperature while being stirred in a disposable spectrophotometercuvette to keep the cells in suspension. After the incubation period,cytosol is separated as the supernatant, following centrifugation at20,800×g in a refrigerated microfuge for 10 minutes at 4° C., from thepellet (which comprises cytosol-depleted cells and containsmitochondria, other organelles and plasma membrane). Thecytosol-depleted, digitonin permeabilized cells are resuspended again inbasal medium, pelleted by centrifugation, and the supernatant fluid isremoved. The pellet containing all cellular organelles (Fiskum et al.,1980) is transferred to a suitable reaction vessel and assayed indigitonin-free incubation medium with respiratory substrates (e.g.,glutamate/malate).

A candidate agent for use according to the present invention may be anycomposition of matter that is suspected of altering mitochondrialfunction as provided herein, in a manner that detectably alters a signalgenerated by a calcium indicator molecule in a cell-based assay asdescribed herein. Detectable alteration of a signal generated by acalcium indicator molecule typically refers to a statisticallysignificant alteration (e.g., increase or decrease) of the signaldetected at at least one of a plurality of time points. According to thepresent invention, a candidate agent that is identified as an agent thatalters mitochondrial function as provided herein cannot be any of thefollowing compounds, although such compounds may be present in any ofthe present screening assays: rotenone, oligomycin, succinate, Bcl-2, orcyclosporin A.

Preferably the candidate agent is provided in soluble form. Withoutwishing to be bound by theory, a candidate agent may directly alter theactivity of a mitochondrial molecular component that regulates cytosolicfree calcium levels, such as a calcium channel or uniporter (e.g., byphysical contact with the calcium channel), or may do so indirectly(e.g., by interaction with one or more additional molecular componentssuch as mitochondrial molecular components present in a sample, wheresuch additional components alter mitochondrial calcium regulatoryactivity in response to contact with the agent). Typically, and inpreferred embodiments such as for high throughput screening, candidateagents are provided as “libraries” or collections of compounds,compositions or molecules. Such molecules typically include compoundsknown in the art as “small molecules” and having molecular weights lessthan 10⁵ daltons, preferably less than 1 daltons and still morepreferably less than 10³ daltons.

For example, members of a library of test compounds can be administeredto a plurality of samples in each of a plurality of reaction vessels ina high throughput screening array as provided herein, each containing atleast one cell containing cytosol, a mitochondrion and a calciumindicator molecule under conditions as provided herein. The samples arecontacted with a source of calcium cations and then assayed for adetectable signal generated by the calcium indicator molecule at aplurality of time points, and the signal generated from each sample inthe presence of the candidate agent is compared to the signal generatedin the absence of the agent. Compounds so identified as capable ofinfluencing mitochondrial function (e.g., alteration of calciumuniporter activity) are valuable for therapeutic and/or diagnosticpurposes, since they permit treatment and/or detection of diseasesassociated with altered mitochondrial function. Such compounds are alsovaluable in research directed to molecular signaling mechanisms thatinvolve a mitochondrial calcium uniporter, and to refinements in thediscovery and development of future uniporter-specific compoundsexhibiting greater specificity.

Candidate agents further may be provided as members of a combinatoriallibrary, which preferably includes synthetic agents prepared accordingto a plurality of predetermined chemical reactions performed in aplurality of reaction vessels. For example, various starting compoundsmay be prepared employing one or more of solid-phase synthesis, recordedrandom mix methodologies and recorded reaction split techniques thatpermit a given constituent to traceably undergo a plurality ofpermutations and/or combinations of reaction conditions. The resultingproducts comprise a library that can be screened followed by iterativeselection and synthesis procedures, such as a synthetic combinatoriallibrary of peptides (see e.g., PCT/US91/08694 and PCT/US91/04666) orother compositions that may include small molecules as provided herein(see e.g., PCT/US94/08542, EP 0774464, U.S. Pat. No. 5,798,035, U.S.Pat. No. 5,789,172, U.S. Pat. No. 5,751,629). Those having ordinaryskill in the art will appreciate that a diverse assortment of suchlibraries may be prepared according to established procedures, andtested using a biological sample according to the present disclosure.

An agent so identified as one that alters (e.g., increases or decreases)mitochondrial function is preferably part of a pharmaceuticalcomposition when used in the methods of the present invention. Thepharmaceutical composition will include at least one of apharmaceutically acceptable carrier, diluent or excipient, in additionto one or more selected agent that alters mitochondrial function and,optionally, other components.

Therapeutic Applications

Agents identified using the above assays may have remedial, therapeutic,palliative, rehabilitative, preventative and/or prophylactic effects onpatients suffering from, or potentially predisposed to developing,diseases and disorders associated with alterations in mitochondrialfunction. Such diseases may be characterized by abnormal, supernormal,inefficient, ineffective or deleterious calcium regulatory activity, forexample, defects in uptake, release, activity, sequestration, transport,metabolism, catabolism, synthesis, storage or processing of calciumand/or directly or indirectly calcium-dependent biological molecules andmacromolecules such as proteins and peptides and their derivatives,carbohydrates and oligosaccharides and their derivatives includingglycoconjugates such as glycoproteins and glycolipids, lipids, nucleicacids and cofactors including ions, mediators, precursors, catabolitesand the like.

Such diseases and disorders include, by way of example and notlimitation, chronic neurodegenerative disorders such as Alzheimer'sdisease (AD) and Parkinson's disease (PD); auto-immune diseases;diabetes mellitus, including Type I and Type II; mitochondria associateddiseases, including but not limited to congenital muscular dystrophywith mitochondrial structural abnormalities, fatal infantile myopathywith severe mtDNA depletion and benign “later-onset” myopathy withmoderate reduction in mtDNA, MELAS (mitochondrial encephalopathy, lacticacidosis, and stroke) and MIDD (mitochondrial diabetes and deafness);MERFF (myoclonic epilepsy ragged red fiber syndrome); arthritis; NARP(Neuropathy; Ataxia; Retinitis Pigmentosa); MNGIE (Myopathy and externalophthalmoplegia; Neuropathy; Gastro-Intestinal; Encephalopathy), LHON(Leber's Hereditary Optic Neuropathy), Kearns-Sayre disease; Pearson'sSyndrome; PEO (Progressive External Ophthalmoplegia); Wolfram syndrome;DIDMOAD (Diabetes Insipidus, Diabetes Mellitus, Optic Atrophy,Deafness); Leigh's Syndrome; dystonia; schizophrenia; andhyperproliferative disorders, such as cancer, tumors and psoriasis.

In contrast to chronic neurodegenerative diseases, neuronal deathfollowing stroke occurs in an acute manner. A vast amount of literaturenow documents the importance of mitochondrial function in neuronal deathfollowing ischemia/reperfusion injury that accompanies stroke, cardiacarrest and traumatic injury to the brain. Experimental support continuesto accumulate for a central role of defective energy metabolism,alteration in mitochondrial function leading to increased oxygen radicalproduction and impaired intracellular calcium homeostasis, and activemitochondrial participation in the apoptotic cascade in the pathogenesisof acute neurodegeneration.

A stroke occurs when a region of the brain loses perfusion and neuronsdie acutely or in a delayed manner as a result of this sudden ischemicevent. Upon cessation of the blood supply to the brain, tissue ATPconcentration drops to negligible levels within minutes. At the core ofthe infarct, lack of mitochondrial ATP production causes loss of ionichomeostasis, leading to osmotic cell lysis and necrotic death. A numberof secondary changes can also contribute to cell death following thedrop in mitochondrial ATP. Cell death in acute neuronal injury radiatesfrom the center of an infarct where neurons die primarily by necrosis tothe penumbra where neurons undergo apoptosis to the periphery where thetissue is still undamaged (Martin et al., Brain Res. Bull. 46:281-309,1998).

Much of the injury to neurons in the penumbra is caused byexcitotoxicity induced by glutamate released during cell lysis at theinfarct focus, especially when exacerbated by bioenergetic failure ofthe mitochondria from oxygen deprivation (MacManus and Linnik, J.Cerebral Blood Flow Metab. 17:815-832, 1997). The initial trigger inexcitotoxicity is the massive influx of Ca²⁺ primarily through the NMDAreceptors, resulting in increased uptake of Ca²⁺ into the mitochondria(reviewed by Dykens, “Free radicals and mitochondrial dysfunction inexcitotoxicity and neurodegenerative diseases” in Cell Death andDiseases of the Nervous System, V. E. Koliatos and R. R. Ratan, eds.,Humana Press, New Jersey, pages 45-68, 1999). The Ca²⁺ overloadcollapses the mitochondrial membrane potential (Δψ_(m)) and inducesincreased production of reactive oxygen species (Dykens, J. Neurochem.63:584-591, 1994; Dykens, “Mitochondrial radical production andmechanisms of oxidative excitotoxicity” in The Oxygen Paradox, K. J. A.Davies, and F. Ursini, eds., Cleup Press, U. of Padova, pages 453-467,1995). If severe enough, Δψ_(m) collapse and mitochondrial Ca²⁺sequestration can induce MPT, indirectly releasing cytochrome c andother proteins that initiate apoptosis (Bernardi et al., J. Biol Chem267:2934-2939, 1994; Zoratti et al., Biochim Biophys Acta 1241:139-176,1995; Ellerby et al., J. Neurosci 17:6165-6178, 1997). Consistent withthese observations, glutamate-induced excitotoxicity can be inhibited bypreventing mitochondrial Ca²⁺ uptake or blocking MPT (Budd et al., J.Neurochem 66:403-411, 1996; White et al., J. Neurosci 16:5688-5697,1996; Li et al., Brain Res 753:133-140, 1997; Stout et al., Nat.Neurosci. 1:366-373, 1998).

Agents and methods that maintain mitochondrial integrity duringischemia/ reperfusion, traumatic tissue injury and/or seizures would beexpected to be novel protective agents with utility in limiting anyischemia/ reperfusion injury to bodily tissues. Given the limitedtherapeutic window for blockade of necrotic death at the core of aninfarct, it may be particularly desirable to develop therapeuticstrategies to limit neuronal death by preventing mitochondrialdysfunction in the non-necrotic regions of an infarct. As providedherein, such agents may be identified by screening collections ofcompounds for their ability to alter (e.g., increase or decrease)mitochondrial regulation of cytosolic calcium under excitotoxicconditions that mimic transient ischemia. Without wishing to be bound bytheory, preferred agents for stroke may be those that lower or reducemitochondrial calcium uptake. Such agents are expected to have remedial,therapeutic, palliative, rehabilitative, preventative, prophylactic ordisease-impeditive effects on patients who have had, or who are thoughtto be predisposed to have, strokes. The cytosolic calcium-based assay ofthe present invention can also be used to estimate which agent(s) aremost likely to be effective for a given individual, in that a patienthaving mitochondria that exhibit altered calcium regulation is expectedto be more likely to respond to agents that modulate mitochondrialregulation of calcium than a patient having mitochondria with a normalcalcium regulatory profile.

Conversely, in certain other disease indication areas, a desiredproperty of an agent that alters mitochondrial function with respect tocalcium regulatory activity may be promotion of calcium uptake orretention by mitochondria. For example, in certain types of cancer, orin certain cells that are transformed with genes known to beoverexpressed in cancer cells, elevated cytosolic calcium levels mayhave deleterious effects that would be potentially overcome bysequestration of excess calcium in mitochondria. Accordingly,identification of agents according to the present invention thatup-regulate mitochondrial uptake may therefore provide beneficialtherapeutic agents. Similarly, in any number of other disease models,cell systems or other biological contexts, for example, in systemswherein cells are identified that are particularly sensitive to stressesfrom inappropriate calcium management (or that can be made so, forinstance, by altering the expression of apoptosis pathway componentssuch as Bcl-2, by exposure to apoptogens or by exposure to agents thatalter intracellular calcium distribution), the present invention offersopportunities to identify agents that alter aberrant calcium regulationby altering mitochondrial function.

“Pharmaceutically acceptable carriers” for therapeutic use are wellknown in the pharmaceutical art, and are described, for example, inRemingtons Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaroedit. 1985). For example, sterile saline and phosphate-buffered salineat physiological pH may be used. Preservatives, stabilizers, dyes andeven flavoring agents may be provided in the pharmaceutical composition.For example, sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid may be added as preservatives. Id. at 1449. In addition,antioxidants and suspending agents may be used. Id

“Pharmaceutically acceptable salt” refers to salts of the compounds ofthe present invention derived from the combination of such compounds andan organic or inorganic acid (acid addition salts) or an organic orinorganic base (base addition salts). The compounds of the presentinvention may be used in either the free base or salt forms, with bothforms being considered as being within the scope of the presentinvention.

The pharmaceutical compositions that contain one or more agents thatalter mitochondrial function as provided herein may be in any form whichallows for the composition to be administered to a patient. For example,the composition may be in the form of a solid, liquid or gas (aerosol).Typical routes of administration include, without limitation, oral,topical, parenteral (e.g., sublingually or buccally), sublingual,rectal, vaginal, and intranasal. The term parenteral as used hereinincludes subcutaneous injections, intravenous, intramuscular,intrasternal, intracavernous, intrameatal, intraurethral injection orinfusion techniques. The pharmaceutical composition is formulated so asto allow the active ingredients contained therein to be bioavailableupon administration of the composition to a patient. Compositions thatwill be administered to a patient take the form of one or more dosageunits, where for example, a tablet may be a single dosage unit, and acontainer of one or more compounds of the invention in aerosol form mayhold a plurality of dosage units.

For oral administration, an excipient and/or binder may be present.Examples are sucrose, kaolin, glycerin, starch dextrins, sodiumalginate, carboxymethylcellulose and ethyl cellulose. Coloring and/orflavoring agents may be present. A coating shell may be employed.

The composition may be in the form of a liquid, e.g., an elixir, syrup,solution, emulsion or suspension. The liquid may be for oraladministration or for delivery by injection, as two examples. Whenintended for oral administration, preferred compositions contain, inaddition to one or more agents that alter mitochondrial function, one ormore of a sweetening agent, preservatives, dye/colorant and flavorenhancer. In a composition intended to be administered by injection, oneor more of a surfactant, preservative, wetting agent, dispersing agent,suspending agent, buffer, stabilizer and isotonic agent may be included.

A liquid pharmaceutical composition as used herein, whether in the formof a solution, suspension or other like form, may include one or more ofthe following adjuvants: sterile diluents such as water for injection,saline solution, preferably physiological saline, Ringer's solution,isotonic sodium chloride, fixed oils such as synthetic mono ordiglycerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The parenteral preparation can be enclosedin ampoules, disposable syringes or multiple dose vials made of glass orplastic. Physiological saline is a preferred adjuvant. An injectablepharmaceutical composition is preferably sterile.

A liquid composition intended for either parenteral or oraladministration should contain an amount of an agent that altersmitochondrial function as provided herein such that a suitable dosagewill be obtained. Typically, this amount is at least 0.01 wt % of theagent in the composition. When intended for oral administration, thisamount may be varied to be between 0.1 and about 70% of the weight ofthe composition. Preferred oral compositions contain between about 4%and about 50% of the agent(s) that alter mitochondrial function.Preferred compositions and preparations are prepared so that aparenteral dosage unit contains between 0.01 to 1% by weight of activecompound.

The pharmaceutical composition may be intended for topicaladministration, in which case the carrier may suitably comprise asolution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, beeswax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device. Topical formulations may contain aconcentration of the agent that alters mitochondrial function of fromabout 0.1 to about 10% w/v (weight per unit volume).

The composition may be intended for rectal administration, in the form,e.g., of a suppository which will melt in the rectum and release thedrug. The composition for rectal administration may contain anoleaginous base as a suitable nonirritating excipient. Such basesinclude, without limitation, lanolin, cocoa butter and polyethyleneglycol. In the methods of the invention, the agent(s) that altermitochondrial function identified as described herein may beadministered through use of insert(s), bead(s), timed-releaseformulation(s), patch(es) or fast-release formulation(s).

It will be evident to those of ordinary skill in the art that theoptimal dosage of the agent(s) that alter mitochondrial function maydepend on the weight and physical condition of the patient; on theseverity and longevity of the physical condition being treated; on theparticular form of the active ingredient, the manner of administrationand the composition employed. It is to be understood that use of anagent that alters mitochondrial function as disclosed herein in achemotherapeutic composition can involve such an agent being bound toanother compound, for example, a monoclonal or polyclonal antibody, aprotein or a liposome, which assist the delivery of said agent.

Species-Specific Agents

In certain embodiments, the present invention provides screening assaysfor identifying species-specific agents. A “species-specific agent”refers to an agent that affects mitochondrial calcium regulation in onesource (e.g., species) but that does not substantially affect themitochondrial calcium regulation in a second source. In other words, theagent should have an effect on one species that is at least twice theeffect on the other species. The screening assays provided herein may beused to identify such agents, using cells and/or mitochondria obtainedfrom different biological sources.

This embodiment of the invention may be used, for example, to identifyagents that selectively induce mitochondrial calcium-mediated apoptosisin different species, e.g., in trypanosomes (Ashkenazi et al., Science281:1305-1308, 1998), and other eukaryotic pathogens and parasites,including but not limited to insects, but which do not induce apoptosisin the cells of their mammalian hosts. Such agents are expected to beuseful for the prophylactic or therapeutic management of such pathogensand parasites. For example, Ridgley et al. (Biochem. J. 340:33-40, 1999)describe a Ca²⁺-dependent cell death process in the unicellular organismTrypanosoma brucei brucei, a parasite of wild gane animals in Africathat is related to the causative agents of “sleeping sickness” inhumans. Following ROS (reactive oxygen species) production,mitochondrial Ca²⁺ transport was impaired and the Ca²⁺ barrier betweenthe nuclear envelope and the cytosol was disrupted. As a consequence,mitochondria were unable to act as “Ca²⁺ reservoirs” and Ca²⁺accumulated in the nucleus, where extensive DNA fragmentation tookplace. Trypanosomes expressing murine Bcl-2 were not protected from ROS(even though Bcl-2-mediated protection from ROS and mitochondrialdysfunction have been reported for mammalian cells). The parasitichaemoflagellate T. brucei brucei thus seems to have, like metazoans, aCa²⁺-dependent cell death pathway which nonetheless has differences(e.g., in terms of its biochemistry) from metazoan Ca²⁺-dependent celldeath pathways. One goal of the present invention is to exploit suchdifferences to find, using the screening assays of the disclosure,compounds that have an antibiotic effect because they induce theTrypanosomal, but not the mammalian, Ca²⁺-dependent cell death pathway.

By way of another example, members of the phylum Apicomplexa (formerlycalled Sporozoa) comprise a large and diverse group of pathogenicprotozoa that are intracellular parasites. Some members, includingspecies of Babesia, Theileria and Eimeria, cause economically importantanimal diseases, and other members, such as Toxoplasma gondii andCryptosporidium spp. also cause human disease, particularly inimmunocompromised individuals. The acomplexicans are unusual in terms oftheir extrachromosomal DNA elements, as they comprise both amitochondrial genome and a putative plastid genome (see Feagin, Annu.Rev. Microbiol. 48:81-104, 1994, for a review). Probably the mostwell-studied acomplexicans are species of Plasmodium, which causemalaria. Antimalarial agents include agents that specifically impact thefunction of Plasmodium mitochondria (Peters et al., Ann. Trop. Med.Parsitol. 78:567-579, 1984; Basco et al., J. Eukaryot. Microbiol.41:179-183, 1994), and one such agent, atovaquone, collapses Δ_(ψ) inmitochondria from Plasmodium yoelii but has no effect on Δ_(ψ) ofmammalian mitochondria (Srivastava et al., J. Biol. Chem. 272:3961-3966,1997). Accordingly, the assays provided herein can be used to screenlibraries of compounds for novel antimalarial agents, such as compoundsthat cause Δ_(ψ collapse in) Plasmodium mitochondria but not inmammalian mitochondria.

As another example, screening methods provided herein may be used toidentify agents that selectively induce Δ_(ψ) collapse in mitochondriaderived from undesirable plants (e.g., weeds) but not in desirableplants (e.g., crops), or in undesirable insects (in particular, membersof the family Lepidoptera and other crop-damaging insects) but not indesirable insects (e.g., bees) or desirable plants. Such agents areexpected to be useful for the management and control of such undesirableplants and insects. Cultured insect cells, including for example, theSf9 and Sf21 cell lines derived from Spodoptera frugiperda, and the HIGHFIVE™ cell line from Trichopolusia ni (these three cell lines areavailable from Invitrogen, Carlsbad, Calif.) may be the source ofmitochondria in certain such embodiments of the invention.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

The following Examples illustrate the invention and are not intended to10 limit the same. Those skilled in the art will recognize, or be ableto ascertain through routine experimentation, numerous equivalents tothe specific substances and procedures described herein. Suchequivalents are considered to be within the scope of the presentinvention.

EXAMPLES

As noted above, a variety of different types of samples containingmitochondria, or derivatives thereof such as submitochondrial particles(SMPs), can be used in the methods of the assay. Whole(nonpermeabealized) cells can be used, but have potential drawbacksrelative to permeabilized cells as explained below.

A permeabilized cell is a cell that has been treated in a manner thatresults in a partial or complete loss of plasma membrane selectivepermeability. As a first example, it may be desirable to permeabilize acell in a manner that permits calcium cations in the extracellularmilieu to diffuse into permeabilized cells and contact mitochondria.Thus, in this instance, permeabilization serves as an alternative to theuse of a calcium ionophore. As a second example, certain detectablemolecules, such as calcium indicator molecules capable of generating adetectable signal as provided herein, may penetrate the plasma membraneat a moderate rate, or to a limited degree, unless their entry into thecytosol is facilitated in some manner. Permeabilization of cells is onemanner by which the cytosolic entry of such detectable molecules can befacilitated. As a third example, some candidate agents being testedaccording to the method may penetrate the plasma membrane at a moderaterate, or to a limited degree, unless their entry into the cytosol isfacilitated in some manner. Perneabilization of cells is one manner bywhich the entry of such candidate agents into the cytosolic space can befacilitated. Active agents that are identified under these conditionscan subsequently be chemically modified to enhance their uptake by wholecells; active agents that are so modified are expected to serve as leadcompounds for drug development and, in some instances, may themselves beused as drugs or as drug candidates.

As also described above, those having ordinary skill in the art arefamiliar with methods for permeabilizing cells, for example by way ofillustration and not limitation, through the use of surfactants,detergents, phospholipids, phospholipid binding proteins, enzymes, viralmembrane fusion proteins and the like; by exposure to certain bacterialtoxins, such as (α-hemolysin); by contact with hemolysins such assaponin (which is also a nonionic detergent, as is digitonin); throughthe use of osmotically active agents; by using chemical crosslinkingagents; by physicochemical methods including electroporation and thelike, or by other permeabilizing methodologies including, e.g., physicalmanipulations such as electroporation. Determination of the mostappropriate permeabilizing agent in a particular context may be based onfactors including toxicity of the agent to a specific chosen cell line,the molecular size of the agent that it is desired to have enter cells,and the like (see, e.g., Schulz, Methods Enzymol. 192:280-300, 1990).

Thus, cells may be permeabilized using any of a variety of knowntechniques, for instance, by the addition of permeabilizing agents suchas the bacterial toxins streptolysin O or Staphylococcus aureus α-toxin(a.k.a. α-hemolysin); other hemolytic agents such as saponin; orexposure to one or more detergents (e.g., digitonin, Triton X-100,NP-40, n-Octyl β-D-glucoside and the like) at concentrations below thoseused to lyse cells and solubilize membranes (i.e., below the criticalmicelle concentration). Certain common transfection reagents, such asDOTAP, may also be used. ATP can also be used to permeabilize intactcells, as may be low concentrations of chemicals commonly used asfixatives (e.g., formaldehyde). All of the permeabilizing agentsdescribed in this paragraph are available from, e.g., Sigma ChemicalCo., St. Louis, Mo. (see Sigma catalog entitled “Biochemicals andReagents for Life Science Research,” Anon., 1999, and references citedtherein for these and other permeabilizing agents).

Permeabilized cells can be assayed for uniporter activity in thepresence and/or absence of compounds of interest (e.g., candidate agentsthat alter mitochondrial functions, including unicouplers of oxidativephosphorylation from ATP production, respiratory inhibitors, or agentsthat alter mitochondrial calcium uniporter activity). Acalcium-sensitive detectable reagent, which may be a fluorophore, ispresent in the media, and conditions are established in which thereagent is free to diffuse into the cytosolic space of permeabilizedcells but does not enter one or more subcellular calcium reservoirs suchas, for example, organelles such as the endoplasmic reticulum (ER) ormitochondria. Ca²⁺ ions also freely enter permeabilized cells; however,in one embodiment of the invention, due to the presence of one or moreintracellular Ca²⁺ modulating agents (ICMAs), Ca²⁺ ions do not enterand/or are released from certain intracellular calcium reservoirs suchas, for example, the ER or mitochondria.

Isolated (i.e., physically separated from the cellular environment inwhich they are naturally present or biosynthesized) or semi-purifiedmitochondria, or submitochondrial particles, may also be used in theassays of the invention. Methods of purifying mitochondria from avariety of species, cells, tissues and organs are known in the art (see,for example, Glick et al., Methods in Enzymology 260:213-223, 1995;Scholte et al., Mol. Cell. Biochem. 174:61-66, 1997; Almeida et al.,Brain Res. 764:167-172, 1997; Schild et al., Acta Opthalmol. Scand74:354-357, 1996). Thus, according to certain embodiments of the presentinvention a biological sample as provided herein may comprise one ormore isolated mitochondria, preferably provided as a suspension in aliquid medium such as a suitable aqueous buffer. More preferably such abuffer is a “respiratory medium” capable of supporting mitochondrialrespiratory activities (e.g., oxidative phosphorylation), and still morepreferably such a respiratory medium permits maintenance ofmitochondrial membrane potential as described herein.

In addition to the methods cited above, preparation of mitochondria incertain preferred embodiments may employ the methods and compositions(including respiratory media) described in Greenawalt et al. (1970 J.Cell Biol. 46:173), Greenawalt (1979 Meths. Enzymol. 55:88) or Pedersonet al. (1978 Meth. Cell Biol. 20:411). Further according to the certainembodiments of the invention wherein the biological sample comprises oneor more isolated mitochondria and a calcium indicator molecule in amedium (such as a respiratory medium), the calcium indicator moleculecapable of generating a detectable signal that is proportional to thelevel of calcium in the sample is preferably an indicator ofintramitochondrial calcium such as Rhod-2 or a derivative thereof (e.g.,Rhod-2-AM), membrane permeable forms of Fura-2 or a derivative thereof(e.g., Fura-2-AM) or aequorin. These and other suitable indicators areavailable from Molecular Probes, Inc. (Eugene, Oreg.) and are describedin Haugland, 1996 Handbook of Fluorescent Probes and ResearchChemicals-Sixth Ed., (Molecular Probes, Eugene, Oreg.) including thereferences cited therein. According to certain other embodiments of theinvention wherein the biological sample comprises one or more isolatedmitochondria and a calcium indicator molecule in a medium (such as arespiratory medium), the calcium indicator molecule capable ofgenerating a detectable signal that is proportional to the level ofcalcium in the sample is preferably an indicator of extramitochondrialcalcium such as Calcium Green™ 5N or a non-membrane permeable form ofFura-2 such as a Fura-2 salt, which indicators are also available fromMolecular Probes, Inc. (Eugene, Oreg.), and which are also, along withother suitable indicators, described in Haugland (1996). As notedelsewhere herein, under certain conditions calcium cation transportacross the mitochondrial inner membrane may be mediated by a knownNa⁺/Ca⁺ antiporter (and/or by a Ca⁺/H⁺ exchanger), albeit at markedlylower kinetic rates than the mitochondrial calcium uniporter. It maytherefore be desirable according to certain embodiments disclosed hereinto perform the subject invention methods in the absence of anyexogenously introduced sodium cations, which according to non-limitingtheory should eliminate or reduce any detectable contribution by theNa⁺/Ca⁺ antiporter to the calcium signal level.

Methods are also known in the art for the preparation ofsubmitochondrial particles (SMPs) from isolated or semi-purifiedmitochondria (see, for example, Walker et al., Methods in Enzymology260:163-190, 1995; and Garlid et al., Methods in Enzymology 260:331-348,1995). Although structurally distinct from the mitochondria from whichthey are isolated, SMPs remain many functions of intact mitochondria(see, for example, Muscari et al., Biochim. Biophys. Acta 1015:200-204,1990; and Sastrasinh et al., Am. J. Physiol. 257:F1050-F1058, 1989).

In certain embodiments of the invention, by using the appropriatecombination of calcium-sensitive detectable reagents and ICMAs, a signalthat corresponds to the uptake or release of Ca²⁺ from a particularintracellular Ca²⁺ reservoir, such as the mitochondrion, is generated.For example, if ICMAs that are used to block uptake and/or promoterelease from the ER and other extramitochondrial Ca²⁺ reservoirs, and acalcium-sensitive detectable reagent that does not enter mitochondria isused, any changes in the concentration of the detectable Ca²⁺ signal isdue to the uptake or release of Ca²⁺ from mitochondria (see FIGS. 1-3).

In addition to mitochondria, known intracellular calcium reservoirsinclude other organelles such as the endoplasmic reticulum (ER). The ERis found in most cell types and is composed of a series of flattenedsheets, tubes and sacs that enclose a large intracellular space. Themembrane of the ER is in structural continuity with the nuclear membraneand extends throughout the cytoplasm. Some functions of the ER includethe synthesis and transport of membrane proteins and lipids. Generallyspeaking, two types of ER may exist in a cell. Smooth ER are generallytubular in shape and are typically devoid of attached ribosomes; onemajor function of smooth ER is lipid metabolism. Rough ER typicallyoccurs as flattened sheets, the cytosolic side of which is usuallyassociated with many active (protein-synthesizing) ribosomes.

Thus, in one embodiment, the invention provides an assay forextramitochondrial calcium and factors, such as the calcium uniporter(CaUP), that influence levels of mitochondrial calcium, and methods ofscreening for agents (e.g., chemical compounds) that affect such factorsor otherwise influence levels of mitochondrial calcium. In thisembodiment, ICMAs (all from Calbiochem, San Diego, Calif., unlessotherwise stated) that prevent the uptake by and/or promote the releaseof calcium from the ER and other extramitochondrial calcium reservoirsare used. Such agents include, by way of example and not limitation, forthe ER, thapsigargin and thapsigaricin.

In another embodiment, the invention provides an assay for calciumexcluded from the ER and factors that influence the level of Ca²⁺ in theER, and methods of screening for agents (e.g., chemical compounds) thatinfluence levels of Ca²⁺ in the ER. In this embodiment, ICMAs (all fromCalbiochem, San Diego, Calif., unless otherwise stated) that prevent theuptake by and/or promote the release of calcium from mitochondria andother calcium reservoirs other than the ER are used; such agentsinclude, by way of example and not limitation, for mitochondria,ruthenium red and Ru-360, and uncouplers such as CCCP and FCCP.

The invention solves the following problems or overcomes the followinglimitations. (1) Total extramitochondrial Ca²⁺ can be measured (2) in areal-time kinetic assay thereof that (3) provides for high throughputscreening (HTS) for compounds that influence the action of endogenousmolecules, or molecules artificially introduced into cells, wherein suchmolecules regulate extramitochondrial calcium levels, either directly orindirectly. The invention provides the following advantages: (A) Assayscan be done under conditions that allow the mitochondrial membranepotential to be the driving force for the calcium uniporter; (B)Conflicting signals from extramitochondrial calcium reservoirs can bereduced or eliminated; (C) Because of (A) and (B), the assays of theinvention provides an assay specific for the mitochondrial calciumuniporter.

The Ca²⁺ uniporter (Ca UP) is driven by the mitochondrial membranepotential (low affinity, high Vmax) and is competitively inhibited bySr²⁺, Mn²⁺, La³⁺; inhibited by Mg²⁺, Ruthenium Red, Ru360; activated byADP and inhibited by ATP; and is responsible for Ca²⁺ sequestration thatstimulates the mitochondrial permeability transition (MPT) Pore. Also,the Ca²⁺ uniporter is clearly implicated in ischemia/reperfusion injuryin a variety of tissues, as well as in the excitotoxic death of neuronsand may play a role in chronic neurodegenerative diseases in which theability of cells to cope with large Ca²⁺ loads has been compromised. TheCaUP has not been cloned.

Technical details relating to the performance of assays according tocertain embodiments of the present invention are described herein, andexamples of suitable conditions may be found, for instance, in Murphy etal. (Proc. Natl. Acad. Sci USA. 93:9893-9898, 1996). In brief, in someembodiments of the invention, cells are permeabilized in the presence ofrespiratory media (potassium chloride-based, containing mitochondrialrespiratory substrates such as glutamate, malate, succinate, pyruvateplus malate, and numerous others) with a calcium-sensitive detectablereagent. This reagent can be, for example, Calcium-Green-5N (MolecularProbes, C-3737, hexapotassium salt) used at 0.1 to 1.0 micromolar. Othercalcium-sensitive detectable reagents, including but not limited tothose presented in Table 1, can be used. The cells are currentlysuspended in the medium, but they could potentially be attached to thesurface of the 96-well plate. For the suspended cell assay, the cellsare at a concentration of ˜1×10⁷ cells/ml, 0.1 ml per well). One skilledin the art can determine appropriate cell concentrations for differentassay reagents, cell lines, instrumentation, etc. TABLE 1CALCIUM-SENSITIVE DETECTABLE REAGENTS Com- Calcium-Sensitive mercialDetectable Reagent Source(s)¹ Notes Arsenazo III Cal useful forspectrophotometric measurements Chlortetracyline Cal fluorescent FF6 &AM ester² Cal fluorescent; properties similar to Fura-2 Fluo-3 & AMester Cal, MP fluoresces upon binding calcium Fluo-3FF & AM ester Calsimilar to Fluo-3, but lower affinity for calcium and insensitive tomagnesium Fura-2 & AM ester Cal, MP fluorescent Fura-2FF & AM ester Calsimilar to Fura-2, but lower affinity for calcium and insensitive tomagnesium Fura-PE3 & AM ester Cal resists rapid leakage &compartmentalization sometimes seen w/Fura-2 Indo-1 & AM ester Cal, MPshift in fluorescence emission (482 → 398 nm) upon Ca²⁺ binding Indo-1FF& AM ester Cal similar to Indo-1, but lower affinity for calcium andinsensitive to magnesium Quin-2 & AM ester Cal, MP Ca²⁺ binding causesmajor shift in uv absorption spectrum Rhod-AM MP loads intomitochondrial matrix Mitochondrially- MP luminescent in the presence ofCa²⁺ directed aequorin when reconstituted with coelenterazine and O₂¹“Cal,” Calbiochem, San Diego, CA; “MP,” Molecular Probes, Eugene, OR;²“AM ester,” acetoxymethyl ester.

The media can contain a chelating agent including but not limited to,BAL (British Anti-Lewisite), DMPS (2,3-dimercapto-1-propanesulfonicacid), EDTA (ethylenediamine-tetraacetic acid), DMSA(meso-2,3-dimercaptosuccinic acid), Penicillamine, DTPA(diethylenepentaacetic acid, Desferrioxamine, DTC (dithiocarbamate), andthe like. The purpose for adding chelating agents to is to removepotentially contaminating Ca²⁺ which may be present in the cellularmedia or in other reagents used in the assays of the invention. At somelevel, contaminating calcium ions could lead to undesirable Ca²⁺sequestration by mitochondria prior to the beginning of the assay; thiswill in turn limit the amount of additional calcium that mitochondriacan sequester. The appropriate concentration of a chelating agent isdetermined empirically, or atomic absorption can be done on media toexamine the amount of calcium present therein, or differentconcentrations of the chelating agent can be added, in the presence of acalcium-sensitive detectable reagent, to the media or reagents, andfluorescent signal corresponding to calcium levels is measured. From theresults of these determinations, it is possible to determine the minimumconcentration of chelating agent beyond which no further changes influorescence can be realized; this concentration of chelating agent isthe minimum required to remove endogenous calcium from media andreagents if need be.

Additionally or alternatively, the media can contain the ICMAthapsigargin to prevent Ca²⁺ uptake and retention by the endoplasmicreticulum (ER).

When Calcium-Green™-5N is used, fluorescence reading may be done inlaser fluorometer such as Molecular devices' FLIPR at 488 um excitation,emission peak at 530±12.5 um. Once baseline fluorescence is established,a known quantity of Ca²⁺ can be added (robotically by the FLIPR from aconcentrated stock in respiratory media), resulting, in the immediateterm, in an increase in signal (fluorescence).

The amount of Ca²⁺ to be added is dependent upon the number ofmitochondria in the assay. In general, the dose of Ca²⁺ should be lowenough so as to not induce opening of the MPT pore (Bernardi et al., J.Biol. Chem. 267:2934-2939, 1992; Bernardi et al., J. Biol. Chem.268:1005-1010, 1993; Szabo et al., J. Biol. Chem. 267:2940-2946, 1992;Zoratti et al., Biochem. Biophys. Acta—Rev. Biomemembranes 1247:139-176,1995).

Typically, a dose response curve for Ca²⁺ is done for a cell line, withthe output being a spontaneous release of Ca²⁺ from Ca²⁺-loadedmitochondria or a transition-like behavior, with Ca²⁺ concentrations of,e.g., 0.1, 0.5, 1.0, 2.5, 5.0, 7.5, 10, 12.5, 15, 20.0, 25, 30, 50, 75and 100 uM. The optimal concentration of Ca²⁺ for a particular cell lineis determined as a concentration well below the maximal Ca²⁺ uptakecapacity as determined by repeated pulses of known quantities of Ca²⁺(Murphy et al., Proc. Natl. Acad Sci. USA 93:9893-9898, 1996). For Hep2(human hepatoma) cells, an appropriate concentration of Ca²⁺ is 6 to 20micromolar CaCl₂ with 1×10 e7 cells/ml in 0.1 milliliter.

As the calcium uniporter functions, mitochondria take up calcium fromthe media, but the calcium-sensitive detectable reagent is not taken upinto mitochondria; the signal thus decreases to basal or near-basallevels. Uniporter activity thus corresponds to a decrease in signal (dueto mitochondrial sequestration of Ca²⁺) following the initial “spike”immediately after calcium is added to the media.

In one form of the assay, following this initial sequestered pulse ofCa²⁺, a test compound is added to the cells before a second Ca²⁺ pulseis applied. A test compound is an agent that is being screened for itsability to influence mitochondrial Ca²⁺ levels in a number of ways (seeFIG. 4).

First, some agents will result in a rise in the signal from thecalcium-sensitive detectable reagent even before the second Ca²⁺ pulseis applied to the cells. For example, an agent that induces MPTuncouples respiration and/or causes a loss in the mitochondrial membranepotential or otherwise gross structural damage to mitochondria, willcause the Ca²⁺ sequestered from the first Ca²⁺ pulse to be rapidlyreleased from mitochondria, causing an immediate rise in the signal fromthe detectable reagent. Similarly, an agent that stimulates the effluxof Ca²⁺ from mitochondria by, for example, altering the activity of amitochondrial divalent cation channel or transporter (including thecalcium uniporter) will result in a rise in signal immediately or soonafter the cells are contacted with the agent. For example, agents thatuncouple respiration dissipate the mitochondrial membrane potential,which acts as the driving force for uptake and retention of Ca²⁺.Moreover, dissipation of delta psi allows the uniporter to function inreverse, allowing Ca²⁺ back out of the mitochondria down itsconcentration and electrical gradients. A respiratory inhibitor wouldbehave similarly, although the increase in signal might not be as rapid.

Second, an agent that specifically influences uniporter activity mayhave little or no effect on the signal immediately after the agent isadded, but will result in a second Ca²⁺ pulse that has a differentsequestration curve than the initial Ca²⁺ pulse (some examples are shownin FIG. 4). For example, a less rapid loss of signal after applicationof the second Ca²⁺ pulse, indicating a less rapid mitochondrialsequestration of Ca²⁺, indicates that the test compound is an agent thatinhibits activity of the calcium uniporter. In the extreme case,application of a sufficient amount of an agent that irreversiblyinhibits the calcium uniporter would result in an increased signal that“plateaus” (i.e., does not decrease) after the second Ca²⁺ pulse. Asanother example, a more rapid loss of signal after application of thesecond Ca²⁺ pulse, indicating a more rapid mitochondrial sequestrationof Ca²⁺, indicates that the test compound is an agent that stimulatesactivity of the calcium uniporter or inhibits Ca²⁺ efflux.

EXAMPLE 1 General Assay Reagent, Other Components and Conditions

Calcium

Calcium chloride (CaCl₂) is commercially available (Sigma, St. Louis,Mo.; C3881). In initial experiments, atomic absorption was done on thestock calcium chloride solution in order to precisely determine itsconcentration. Autoclaved stock solutions of 0.025 mol/L are available(Sigma, St. Louis, Mo.). The concentration of CaCl₂ is important becausea difference between 4 and 8 micromolar can be important with regard toinducing spontaneous Ca²⁺ release from mitochondria.

Calcium-Sensitive Detectable Reagents

Calcium-Green-5N (potassium salt) is commercially available (MolecularProbes, Eugene, Oreg.; C-3737). Calcium-Green-5N is a low affinity Ca²⁺indicator (as is, for example, Oregon Green 488 BAPTA-5N). Low affinityindicators are preferred because of the Ca²⁺ concentrations used in theassays. High affinity dyes require a lower Ca²⁺ concentration andtherefore a lower number of cells, and thus a lower number ofmitochondria, would be required than the number used in the assays.Calcium-Green-5N can be frozen and thawed a limited number of timesbefore it loses potency (as demonstrated by a decrease in the startingfluorescence and diminishing peak values). The stock is thus typicallydivided into aliquots that go through 10 or fewer freeze/thaw cyclesbefore being discarded.

Other calcium-sensitive detectable reagents that can be used in theassay of the invention include Calcein, Calcein Blue, Calcium-Green-1,Calcium-Green-2, Calcium-Green-C₁₈, Calcium Orange, Calcium-Orange-5N,Calcium Crimson, Fluo-3, Fluo-3 AM ester, Fluo-4, Fura-2, Fura-2FF, FuraRed™, Fura-C₁₈, Indo-1, Bis-Fura-2, Mag-Fura-2, Mag-Fura-5, Mag-Indo-1,Magnesium Green™, Quin-2, Quin-2 AM (acetoxymethyl) ester, MethoxyquinMF, Methoxyquin MF AM ester, Rhod-2, Rhod-2 AM ester, Texas Red®-CalciumGreen™, Oregon Green™ 488 BAPTA-1, Oregon Green™ 488 BAPTA-2, BTC, BTCAM ester, (all from Molecular Probes, Inc., Eugene, Oreg.), and aequorin(Molecular Probes).

Permeabilizing Agents and Methods

Those having ordinary skill in the art are familiar with methods forpermeabilizing cells, for example by way of illustration and notlimitation, through the use of surfactants, detergents, phospholipids,phospholipid binding proteins, enzymes, viral membrane fusion proteinsand the like; by exposure to certain bacterial toxins, such as(α-hemolysin); by contact with hemolysins such as saponin (which is alsoa nonionic detergent, as is digitonin); through the use of osmoticallyactive agents; by using chemical crosslinking agents; by physicochemicalmethods including electroporation and the like, or by otherpermeabilizing methodologies including, e.g., physical manipulationssuch as, e.g., electroporation. Those skilled in the art familiar withmethods for permeabilizing cells will be able to determine the mostappropriate permeabilizing agent based on factors including but notlimited to toxicity of the agent to a specific chosen cell line, themolecular size of the agent that it is desired to have enter cells, andthe like (see, e.g., Schulz, Methods Enzymol. 192:280-300, 1990).

Thus, for instance, cells may be permeabilized using any of a variety ofknown techniques, such as the addition of permeabilizing agents suchbacterial toxins such as streptolysin O, Staphylococcus aureus α-toxin(a.k.a. a-hemolysin); other hemolytic agents such as saponin; orexposure to one or more detergents (e.g., digitonin, Triton X-100,NP-40, n-Octyl β-D-glucoside and the like) at concentrations below thoseused to lyse cells and solubilize membranes (i.e., below the criticalmicelle concentration). Certain common transfection reagents, such asDOTAP, may also be used. ATP can also be used to permeabilize intactcells, as may be low concentrations of chemicals commonly used asfixatives (e.g., formaldehyde). All of the permeabilizing agentsdescribed in this paragraph are available from, e.g., Sigma ChemicalCo., St. Louis, Mo. (see Sigma catalog entitled “Biochemicals andReagents for Life Science Research,” Anon., 1999, and references citedtherein for these and other permeabilizing agents). In many of theexperiments described herein, digitonin (digitin) was used a cellpermeabilizing agent. Typically, a 10% stock solution in DMSO was madeand stored frozen. Table 2 describes optimal concentrations of digitoninfor permeabilizing several cell lines. TABLE 2 OPTIMAL DIGITONINCONCENTRATIONS FOR SEVERAL CELL LINES Optimal Digitonin Concentrationfor Cell Line Calcium-Green-5N Assay SH-SY5Y (suspended at 1 × 10⁶0.007% cells/well in 0.1 ml) HepG2 (suspended at 1 × 10⁶ cells/well0.007% in 0.1 ml) MixCon (adherent; 6 × 10⁵ cells/well for  0.03% 48hours) Cybrid 1685  0.03%Chelating Agents

The assays of the invention can optionally comprise a chelating agent,particularly if supplies or reagents (such as, e.g., H₂O) needed toprepare components for the assay that are or may be contaminated byCa²⁺. EGTA (ethylene glycol bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid) is commercially available (Sigma, StLouis. Mo.). EDTA is another agent that can be used to chelate Ca²⁺,although it also chelates Mg²⁺. Other calcium chelators can be usedincluding 1-10 phenanthrolene. A 250 millimolar stock solution of EGTA(pH adjusted to 7.0 with KOH) was typically used in experimentsdescribed herein. Optimal concentration of EGTA, if found to benecessary in a given instance, may be influenced by the amount ofcontaminating Ca²⁺ in a given laboratory supply. It may thus benecessary to do a dose response with both EGTA and the Ca²⁺ pulse size.The pulse size of Ca²⁺ used in the methods of the present invention, asdescribed in, inter alia, FIG. 4, depends on factors such asmitochondrial number and mass, but the range of pulse size can be fromone to hundreds of micromolar depending on the particular cell line usedin the assay. Mitochondria prepared from different tissues will toleratedifferent concentrations of Ca²⁺.

Extramitochondrial Calcium Releasing Agents

Thapsigargin is a Ca²⁺ uptake inhibitor of the endoplasmic reticulum(ER) and is commercially available (Calbiochem, San Diego, Calif.).Other agents that release Ca²⁺ from extramitochondrial reservoirs,and/or prevent the uptake of Ca²⁺ into such extramitochondrialreservoirs, include without limitation inositol-1,4,5-triphosphate(Streb et al., Nature 306:67-69, 1983; Berridge et al., FASEB J.2:3074-3082, 1988), okadaic acid (Hepworth et al., Cell Calcium21:461-467, 1997), and caffeine.

Agents that Influence Mitochondrial Functions

Ruthenium Red is a cytological stain that inhibits the Ca²⁺ uniporterand therefore uptake of Ca²⁺ into mitochondria (Reed and Bygrave, Bioch.J. 140:143-155, 1974). It also blocks release of Ca²⁺ from thesarcoplasmic reticulum (Antonius et al., Biochem. Biophys. Acta816:9-17, 1985; Chiesi et al., Biiochem. Biophys. Res. Commun. 154:1-8,1988) and the sequestering ability of the endoplasmic reticulum (Hurley,Am. J. Physiol. 23:621-627, 1988). Ru 360 (Calbiochem, San Diego,Calif.; 557440) is the dinuclear ruthenium amid portion of RutheniumRed, proposed to be responsible for the inhibition effects of RutheniumRed (Ying et al., Biochemistry 30:4949-4952, 1991; Emerson et al., J.Am. Chem. Soc. 115:11799-11805, 1993).

FCCP (carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone; Sigma) is apotent uncoupler of oxidative phosphorylation in mitochondria (Heytleret al., Biophys. Res. Commun. 7:272, 1962; Biochem. J. 195:583, 1981).Other non-limiting examples of respiratory uncouplers include carbonylcyanide m-chlorophenyl-hydrazone (Sigma) (Heytler et al., Biophys. Res.Commun. 7:272-etc., 1962), and those described by Heytler in, e.g.,Methods of Enzymology 55:462, 1979, and Pharmacol. Ther. 10:461-472,1980, both of which are hereby incorporated by reference.

Rotenone (Sigma) is an inhibitor of mitochondrial electron transport(Fukami et al., Science 155:713-716, 1967). Other non-limiting examplesof inhibitors of mitochondrial ETC include cyanide, amytal andantimycin.

Oligomycin (Sigma) is an inhibitor of mitochondrial ATPase (Nagamune etal., Biochim. Biophys. Acta 1141:231-237, 1993). The combination ofoligomycin and rotenone, or rotenone alone, can be used as a positivecontrol to evaluate the effects of greatly reducing or essentiallyelimination of the membrane potential.

Ethacrynic acid (2,3-dichloro4-(2methylene-butryl)phenoxylacetic acid;Sigma) removes the ability of cells to tolerate oxidative stress.Ethacrynic acid inhibits gluatathione S-transferase and thus depletescells of glutathione (Shen et al., Biochem. Pharmacol. 50:1233-1238,1995). Experiments suggest that mitochondrially localized glutathionehas a critical role in the maintenance of mitochondrial function(Seyfried et al., Neurosci. Lett. 264:1-4, 1999).

Apoptogens

In certain aspects of the invention, an altered mitochondrial state isinduced by exposing a biological sample to compositions known as“apoptogens,” agents that induce programmed cell death (PCD or“apoptosis”). For reviews of apoptosis, see Green et al. (Science281:1309-1312, 1998), Raff (Nature 396:119-122, 1998), and Susin et al.(Biochim. et. Biophys. Acta 1366:151-165, 1998).

A variety of apoptogens are known to those familiar with the art and mayinclude by way of illustration herbimycin A (Mancini et al., J. Cell.Biol. 138:449-469, 1997); paraquat (Costantini et al., Toxicology99:1-2, 1995); ethylene glycols(http://www.ulaval.ca/vrr/rech/Proj/532866.html); protein kinaseinhibitors such as, e.g.: staurosporine, calphostin C, caffeic acidphenethyl ester, chelerythrine chloride,1-(5-isoquinolinesulfonyl)-2-methylpiperazine,N-[2-((p-bromocinnamyl)amino)ethyl]-5-5-isoquinolinesulfonamide, KN-93,genistein, quercitin and d-erythro-sphingosine derivatives; ultravioletradiation; ionophores such as, e.g., ionomycin, valinomycin and otherionophores known in the art; MAP kinase inducers such as, e.g.,anisomycin and anandamine; cell cycle blockers such as, e.g.aphidicolin, colcemid, 5-fluorouracil and homoharringtonine;acetylcholineesterase inhibitors such as, e.g., berberine;anti-estrogens such as, e.g., tamoxifen; pro-oxidants such as, e.g.,tert-butyl peroxide and hydrogen peroxide; free radicals such as, e.g.,nitrous oxide; inorganic metal ions, such as, e.g., cadmium; DNAsynthesis inhibitors such as, e.g., actinomycin D, bleomycin sulfate,hydroxyurea, methotrexate, mitomycin C, camptothecin, daunorubicin andDNA intercalators such as, e.g., doxorubicin; protein synthesisinhibitors such as, e.g., cycloheximide, puromycin, and rapamycin;agents that effect microtubule formation or stability such as, e.g.:vinblastine, vincristine, coichicine, 4-hydroxyphenylretinamide andpaclitaxel; gangliosides such as GD3 (Scorrano et al., J. Biol. Chem.274:22581-22585, 1999); agents that may be contacted with cells havingappropriate receptors including, by way of example and not limitation,tumor necrosis factor (TNF), FasL, glutamate, NMDA (the preceding arecontacted with cells having receptors that mediate the uptake of theindicated agent), corticosterone [with cells having mineral corticoid orglucocorticoid receptor(s)]; agents that are withdrawn from the culturemedia of cells after some period of time such as, by way of non-limitingexample, the withdrawal of IL-2 from lymphocytes; and agents that can becontacted with isolated mitochondria or permeabilized, cells including,by way of example and not limitation, calcium and inorganic phosphate,(Kroemer et al., Ann. Rev. Physiol. 60:619-642, 1998) and members of theBax/Bcl-2 family of proteins (Jurgenmeier et al., Proc. Natl. Acad. Sci.U.S.A. 95:4997-5002, 1998). Such agents are prepared according tomethods known in the art or are commercially available from companiessuch as, for example, Calbiochem (San Diego, Calif.) and Sigma ChemicalCompany (St. Louis, Mo.).

Cell Lines

A variety of cells or cell lines or mitochondria isolated or preparedtherefrom, can be used in the present invention. Preferably non-yeasteukaryotic cells, particularly differentiated or undifferentiated cellsor stem cells of endodermic, ectodermic or mesodermic origin. Preferredexamples include, but are not limited to, the following.

“SH-SY5Y” is a line of neuroblastoma cells (Biedler et al., Cancer Res.,38:3751-3757, 1978; see also U.S. Pat. No. 5,888,498, Davis et al.,issued Mar. 30, 1999, hereby incorporated by reference).

Cybrids are hybrid cells that include the nuclear genome of a cell andthe mitochondria from platelets (U.S. Pat. No. 5,888,498).

“MixCon” refers to a mixture of cybrids derived from multiple patients.(U.S. Pat. No. 5,888,498).

“1685 cybrid” refers to a cybrid cell line that combines the nuclearbackground of the SH-SY5Y cell line with mitochondria prepared fromplatelets prepared from a patient tentatively diagnosed with Alzheimer'sDisease. See copending U.S. patent application No. 60/124,673, herebyincorporated by reference.

“L6” is a cell line derived from skeletal muscle myoblasts from rat(Proc. Natl. Acad Sci. USA 61:477-483, 1968; Dev. Biol. 23:1-22, 1970;ATCC CRL-1458).

“293” is a human kidney cell line that has been transformed with thetransforming gene of adenovirus 5 (ATCC CRL-1573, 45504 and 45505).

“HEPG2” is a hepatocellular carcinoma, human (U.S. Pat. No. 4,393,133;Aden et al., Nature (Lond.) 282:615-616, 1979; ATCC HB-8065).

“Jurkat” refers to Jurkat, Clone E6-1, acute T cell leukemia from human(ATCC TIB-152).

Media

Standard cell media were typically from commercial sources as indicatedwith the Examples. The formula for Basic Respiratory Media (a.k.a.“BReM”) is described in Table 3. TABLE 3 BASIC RESPIRATORY MEDIA (BREM)Component (Attribute) Concentration (Value) KCl 125 mM K₂HPO₄  2 mMHepes  20 mM (pH) (adjusted to 7.0 at ambient temperature using KOH)

In experiments wherein the respiratory media contains glutamate, malate,succinate, MgCl₂, Calcium-Green-5N, these additional components areadded on the day of the assay. With the exception of Calcium-Green-5N,stock solutions of these additives are stable to freezing and thawing.In contrast, Calcium-Green-5N is a salt that degrades over time (about amonth), even if stored at −80° C., and that multiple rounds of freezingand thawing enhances this degradation.

Instrumentation

Depending on the assay, a Fluorometric Imaging Plate Reader (FLIPR™)instrument (Molecular Devices, Sunnyvale, Calif.) is often theinstrument of choice for the assays of the invention. The FLIPR™ has thefollowing desirable features (seehttp://www.moleculardevices.com/pages/flipr.html): it uses a combinationof a water-cooled, argon-ion laser illumination and cooled CCD camera asan integrating detector that accumulates signal over the period of timein which it is exposed to the image and, as a result, itssignal-to-noise characteristics are generally superior to those ofconventional imaging optics; it also makes use of a proprietarycell-layer isolation optics that allow signal discrimination on a cellmonolayer, thus reducing undesirable extracellular backgroundfluorescence; it provides data in real-time, and can also providekinetic data (i.e., readings at a multitude of timepoints); it has theability to simultaneously stimulate and read all 96 wells of a 96-wellmicroplate; it provides for precise control of temperature and humidityof samples during analysis; it includes an integrated state-of-the-art96-well pipettor, which uses dispensible tips to eliminate carryoverbetween experiments, that can be used to aspirate, dispense and mixprecise volumes of fluids from microplates; and, in the case of theFLIPR³⁸⁴ instrument, it can be adapted to run sample assays in a roboticor semi-robotic fashion, thus providing for analysis of large numbers ofsamples in shortest amount of time (e.g., up to about a hundred 96-wellmicroplates per day).

EXAMPLE 2 General Assay Protocols

A master mix solution (MM Solution) was prepared by the addition of 5glutamate and malate to final concentrations of 5 millimolar each, MgCl₂to a final concentration of 1 millimolar, EGTA to a final concentrationsof 0 to 8 micromolar, and Calcium-Green-5N to a final concentration of0.1 to 1.0 micromolar, to a basic KCl-based respiratory media (“BReM”)that is described in Table 3. Alternative respiratory media can beprepared using sucrose and/or mannitol with or without phosphate buffer.The hexapotassiun salt Calcium-Green-5N (Molecular Probes, Eugene,Oreg.) in the MM Solution is a fluorescent dye that has low bindingaffinity to Ca²⁺.

Thapsigargin (Calbiochem, San Diego, Calif.), a Ca²⁺ uptake inhibitor ofthe endoplasmic reticulum (ER), was added to the MM Solution to a finalconcentration of 1 micromolar to yield a solution designated “MM-T”(ie., Master Mix with Thapsigargin).

The cell membrane permeabilizing agent digitonin (Sigma, St. Louis, MO)was added to MM-T to a final concentration of from about 0.007% to about0.03% to yield a solution referred to as “MM-TD” (i.e., Master Mix withThapsigargin and digitonin. CaCl₂ was added to MM-T to provide asolution with a determined final concentration of Ca²⁺ for thedetermination of intracellular Ca²⁺ flux.

The range of appropriate digitonin concentrations for different celllines varies and is titrated for each cell line as the cholesterolcontent of the plasma membrane varies with cell type. Thereforedifferent quantities of digitonin are required to selectivelypermeabilize the plasma membrane of the different cell lines. If toolittle digitonin is used, the cells will not be sufficientlypermeabilized to allow access of the dye and reagents to theextramitochondrial space. When too much digitonin is used, the outermitochondrial membrane will become permeabilized and consequentlyrelease factors such as, for example, cytochrome c, thereby limitingrespiration.

Titration of the optimal digitonin concentration for a given cell linecan be accomplished by a variety of methods. For example cells can besuspended in an oxygen electrode chamber in BReM in the presence ofsuccinate and the complex I inhibitor rotenone. Digitonin is titrateduntil a maximal rate of oxygen consumption is reached, therebydetermining the optimal concentration of digitonin. In this approach,succinate does not readily cross the plasma membrane. Therefore, optimalpermeabilization of the plasma membrane provides optimal levels ofoxidizable substrate to the mitochondria.

Alternately the uptake of TTP⁺ (tetraphenyl phosphonium ion) into themitochondrial matrix can be monitored. TTP⁺ is a cation that permeatesthe mitochondrial inner membrane and distributes across the membrane ina Nernstian manner and its distribution reflects the membrane potential.The TTP⁺ concentration in the media is monitored using a TTP⁺-selectiveelectrode. TTP⁺ does not readily cross the plasma membrane, therefore asthe plasma membrane is titrated with digitonin the TTP⁺ gains access tothe mitochondria and is taken up as a function of the membranepotential. The maximal disappearance of TTP⁺ from the medium isindicative of optimal digitonin concentration. Another, but lesssensitive approach to titrating digitonin for an adherent cell line,involves the addition of basic respiratory media (BReM) containing fromabout 0.04% to about to 0.4% trypan blue to the cells, or a fluorescenceplasma membrane impermeant dye such as propidium iodide (5micrograms/milliliter), with increasing concentrations of digitonin. Bylight microscopy, or fluorescence microscopy when using propidiumiodide, the minimal concentration of digitonin that permeabilizes 100%of the cells can be determined. For trypan blue essentially all of thecells become blue and for propidium iodide essentially all of the cells'nuclei become fluorescent. Table 2 describes results for optimization ofdigitonin for several different cell lines.

EXAMPLE 3 Detection of Ca²⁺ Uniporter Activity

Assays were performed to optimize the Ca²⁺ concentration whereby theconcentration allows for Ca²⁺ uniporter transport into mitochondria butis not high enough to induce permeability transition. Stock solutions(10×) of Ca²⁺ were prepared by addition of CaCl₂ to MM-T at finalconcentrations of 0, 40, 80, 120, 160, and 200 micromolar Ca²⁺. Thecells used in this experiment were a mixture of control cybrids (MixCon)from multiple normal individuals (see U.S. Pat. No. 5,888,498). Cellswere trypsinized in Dulbecco's modified Eagle's medium with 10% heatinactivated fetal bovine serum (FBS) and added to wells of a 96-wellCostar 3603 microplate at a concentration of about 6×10⁴ cells in 100microliters per well 48 hours prior to performing assays. Prior to useof the cells, the growth media was aspirated from the wells. One hundredmicroliters of MM-TD was added to each well to permeabilize the cells.The plate was placed into a Fluorometric Imaging Plate Reader (FLIPR™;Molecular Devices Corporation, Sunnyvale, Calif.), heated to 37° C. andilluminated with a 488 nm excitation wavelength. Each of the 10× Castock solutions (i.e., 0, 40, 80, 120 and 160 micromolar Ca²⁺, weredispensed in volumes of 11.1 microiliters into wells containing thepermeabilized cells to yield test samples having Ca²⁺ concentrations of0, 4, 8, 12, and 16 micromolar, respectively.

As intracellular Ca²⁺ is bound to Calcium-Green-5N, the 53.1 nm emissionincreases. As intracellular Ca²⁺ ions cease to be bound to thecalcium-sensitive detectable reagent due to their transport intoorganelles of cells, the resulting emission signal decreases, resultingin a Ca²⁺ ‘spike’ as monitored in the FLIPR™ (FIG. 4, panel A).Thapsigargin is present to inhibit the uptake of Ca²⁺ into the ER andtherefore decreasing cytosolic concentrations of Ca²⁺ in the cytosolreflect the sequesteration of Ca²⁺ in mitochondria. Two additionalinjections with the 10× CaMM solutions at 3 minute intervals in volumesof 12.3 and 13.7 microliters were performed by the FLIPR system. Theresults of these assays are shown in FIG. 5, from which it wasdetermined that the appropriate Ca²⁺ pulse size for MixCon cells rangesfrom about 4 to about 8 micromolar.

EXAMPLE 4 Detection of Modulation of Ca²⁺ Uniporter Activity

A. General Methods

The effects of several intracellular Ca²⁺ modulating agents (ICMAs) onpermeabilized HepG2 cells were examined as follows. The mitochondrialrespiratory inhibitors oligomycin (Calbiochem, San Diego, Calif.) androtenone (Calbiochem) were added to MM-T to final concentrations of 50micrograms/milliliter and 20 micromolar, respectively. The uncouplerFCCP was similarly added to MM-T to a 10× concentration (10 micromolar)to yield a stock solution of FCCP, MM-TF (i.e., Master Mix withThapsigargin and FCCP). The ICMA Ru360, which blocks Ca²⁺ uptake intothe mitochondria by the calcium uniporter, was added to MM-T to aconcentration of 100 micromolar to yield MM-T360 (i.e., Master Mix withThapsigragin and Ru360). CaCl₂ was added to MM-T to yield a 10× solutionof CaCl₂ (120 micromolar) in MM-T.

HepG2 cells were cultured in Eagle's minimal essential medium (MEM) withnon-essential amino acids, sodium pyruvate and Earle's balanced saltsolution (BSS), 90%; fetal bovine serum (FBS), 10%. Cells werecentrifuged (500×g) and the growth media was aspirated from the pellet.The HepG2 cells were resuspended in MM-TD at a concentration of 1×10⁷cells/milliliter, and 100 microliters of the resuspended permeabilizedcells were dispensed into wells of a 96-well Costar 3603 microplate. Theplate was placed into a FLIPR™ prewarmed to 37° C. MM-T, 120 micromolarCa²⁺, was dispensed into the cell-containing wells at a volume of 11.1microliters, yielding a final assay concentration of 12 micromolar Ca²⁺.The addition of Ca²⁺ at this stage of the assay is referred to as thefirst calcium pulse and the time at which Ca²⁺ is added is designatedt=0.

After about three minutes (ie., at about t3 min.), 12.3 microliters ofone of the ICMA solutions were dispensed into wells containingpermeabilized cells to yield final assay concentrations of 5micrograms/milliliter oligomycin, 2 micromolar rotenone, 1 micromolarFCCP, 1 micromolar RU360, and MM-T (no ICMA).

Cells were exposed to a second calcium pulse by the addition of 13.7microliters of MM-T, 120 micromolar Ca²⁺, to the cells after anadditional 3 minutes (i.e., at about t=6 min.). The oxidativephosphorylation inhibitors oligomycin and rotenone, as well as theuncoupler FCCP, caused the release of Ca²⁺ from mitochondria into thecytosol where the Ca²⁺ associated with the Ca²⁺ indicator and theincrease in fluorescence was detected by the FLIPR. Upon addition of athe second Ca²⁺ pulse the mitochondria could not sequester the addedCa²⁺ in these cells. The cells in the presence of Ru360 prevented theuptake of Ca²⁺ by the Ca Uniporter. This is reflected in increasedlevels of Ca²⁺ associated with the calcium indicator in the cytosol.Cells assayed in absence of the ICMA show a decrease in Ca²⁺ associatedwith the Ca²⁺ indicator after each Ca²⁺ pulse and reflects the uptake ofCa²⁺ into the mitochondria

B. Methods Using Adherent Cells

Optimization of the Ca²⁺ response curve and time course of Ca²⁺ pulsesby the FLIPR™ using Calcium-Green-5N was performed in an adherent cellline. The cell line used was MixCon (see Example 1). CaCl₂ was added toMM-T to 10× final concentrations of 0, 40, 80, 100 and 200 micromolar.Immediately prior to the assay, 100 microliters (2×10⁶ cells/milliliter)of trypsinized MixCon cells in Dulbecco's modified Eagle's medium and10% FBS growth media were transferred to wells of a 96-well Costar 3603plate. Wells were coated with Cell-TAK essentially according to themanufacturer's (Becton Dickinson) instructions. The cells werecentrifuged onto the plate and the growth media aspirated from thewells, leaving a layer of cells in each well. Cells were permeabilizedwith the addition of 80 microliters of MM-TD to each well. The digitoninin MM-TD permeabilizes the cells and the thapsigargin in MM-TD inhibitsCa²⁺ uptake by the ER.

Plates were placed in the FLIPR™, and three doses of different 5× stocksolutions of Ca²⁺ in MM-T solutions of 0, 40, 80, 100 and 200 micromolarcalcium in progressive volumes of 20, 25, and 30 microliters weredispensed into the wells at approximately 2 minute intervals so thatconcentrations of Ca²⁺ remained at constant levels of 0, 4, 10 or 20micromolar. Fluorescent emission was monitored at the appropriatewavelength was monitored and cytosolic Ca²⁺ determined throughout assayby the association of Ca²⁺ with the Ca²⁺ indicator. The results with theadherent cell line is presented in FIG. 6.

Mitochondrial inhibitors of oxidative phosphorylation, oligomycin androtenone, were used to observe the effect of decreased membranepotential of mitochondria on cytosol Ca²⁺ levels in the adherent cellline MixCon. The adherent MixCon cells in Dulbecco's modified Eagle'smedium and 10% FBS growth media were dispensed into wells of a 96-wellmicrotiter plate at a concentration of about 6×10⁴ cells in 100microliters per-well 48 hours prior to assay. Just prior to their usethe growth media was aspirated from the wells. Ninety (90) microlitersof MM-TD with oligomycin (5 micrograms/milliliter) or rotenone (2micromolar), or MM-TD without either inhibitor, were added to the cells.The plate was placed in a FLIPR™ system, and three doses of the 10×stock (100 micromolar) solution of calcium were dispensed into thedifferent wells at approximately 2 minute intervals with increasingvolumes (10, 11.1, and 12.3 microliters respectively), resulting in aconstant concentration of 10 micomolar Ca²⁺ throughout the course of theassay. Emissions at the 531 nm wavelength were monitored by a FLIPR™ asa measure of extramitochondrial Ca²⁺-Green-5N fluorescence. The signalcorresponding to extramitochondrial calcium increased after each pulseand, in samples when remained high when in the presence of the twoinhibitors. Alternatively, Ca²⁺ is sequestered in mitochondria of cellsnot exposed to inhibitors (FIG. 7).

Another assay was performed to examine the effect by the respiratoryinhibitors oligomycin and rotenone and subsequent release of Ca²⁺ frommitochondria in the presence or absence of succinate in MixCon cells.Two different master mixes (MM and MM-GMS) were prepared with MMprepared as previously described. MM-GMS was prepared by the addition ofglutamate, malate and succinate to final concentrations of 5 millimolareach, MgCl₂ to a final concentration of 1 millimolar, EGTA to a finalconcentration of 10 micromolar, and Calcium-Green-5N to a finalconcentration of 0.1 micromolar, to a basic KCl-based respiratory media(BReM, Table 3). Thapsigargin was added to the MM and MM-GMS to a finalconcentration of 1 micromolar to yield MM-T and MM-GMS-T (where “T”indicates Thapsigargin and “GMS” indicates glutamate, malate andsuccinate. The cell permeabilizing agent digitonin was added to MM-T andMM-GMS-T to a final concentration of 0.03% to yield solutions designatedas MM-TD and MM-GMS-TD. As indicated in some instances, themitochondrial inhibitors oligomycin and rotenone (Calbiochem) were eachadded to solutions of MM-TD and MM-GMS-TD to final concentrations of 5micrograms/milliliter and 2 micromolar, respectively.

CaCl₂ was added to MM-T and MM-GMS-T to a concentration of 50 micromolarCa²⁺ to yield 5×Ca²⁺ solutions in MM-T and MM-GMS-T, respectively.Versions of the 5×Ca²⁺ stock solutions comprising respiratory inhibitorswere also prepared (oligomycin, final concentration of 50micrograms/milliliter; rotenone, 20 micromolar).

The MixCon cells, in Dulbecco's modified Eagle's medium and 10% FBSgrowth media, were dispensed into wells of a 96-well Costar 3603microtiter plate at a concentration of 6×10⁴ cells in one hundredmicroliters per well, 48 hours prior to the assay. Just prior to theiruse the growth media was aspirated from the wells. Eighty (80)microliters of MM-TD, MM-GMS-TD, MM-TD-O, MM-TD-R, and MM-GMS-TD-OR(where “O” oligomycin and “R” signifies rotenone) were dispensed intowells containing the cells. The microtiter plate was placed in a FLIPR™preheated to 37° C. and an initial injection of 20 microliters of the 5×stock solutions (50 micromolar) of Ca²⁺, with olygomycin, rotenone or noadditions, were dispensed into the appropriate wells such that the Ca²⁺concentration in the assay is 10 micromolar and wells with theinhibitors remain at constant assay concentrations.

A second pulse of 25 microliters of the 5× solution was supplied afterapproximately 5 minutes. The emission wavelength was monitored andintracellular Ca²⁺ determined throughout assay in the FLIPR System.Adherent cells subjected to oligomycin and rotenone in the absence ofsuccinate displayed elevated levels of Ca²⁺-5N fluorescence after thesecond Ca²⁺ pulse indicative of respiratory inhibition and thereforeinhibition of uniporter activity. Alternatively, adherent cellssubjected to oligomycin and rotenone in the presence of succinate show areduction of Ca²⁺-5N fluorescence after the second Ca²⁺ pulse indicatingthat succinate interferes with the respiratory inhibitors by energizingthe mitochondria and providing the driving force for uniporter activity(FIG. 8).

C. Methods Using Suspended Cells

A series of functional assays were performed on non-adherent cell linesin order to optimize the mitochondria Ca-UP assay on FLIPR usingCalcium-Green-5N and digitonin permeabilized cells. A dose response ofCa²⁺ pulses with and without the addition of mitochondria oxidativerespiration uncouplers were performed utilizing cells from HepG2, a cellline derived from a human hepatocellular carcinoma. MM with thapsigarin,MM-T, and digitonin, MM-TD, are prepared as previously described. Tentimes (10×) Ca²⁺ media is prepared by adding CaCl₂ (160 micromolar) toMM-T. The L6 cells were suspended in the cell permeabilizing respiratorymedia MM-TD at a concentration of 1×10⁷ cells per milliliter. Onehundred microliters of the suspension were added to each well of a96-well CoStar 3603 microtiter plate. Digitonin (“D”) in MM-TDpermeabilizes cells and thapsigargin (“T”) in MM-TD inhibits Ca²⁺ uptakeby the endoplasmic reticulum (ER). The plates was placed in the aFLIPR™, heated to 37° C.; then, 11.1 microliters of a stock calciumsolution (160 micromolar CaCl₂ in MM-TD) were dispensed into the 96different wells containing the L6 cells, resulting in an assayconcentration of 16 micromolar of Ca²⁺. After about 3 minutes a seconddose (12.5 microliters) of the MM-TD, 160 micromolar CaCl₂ solution wasdispensed into the wells, thereby maintaining the concentration of Ca²⁺throughout the assay at 16 micromolar. Emissions resulting from[Ca²⁺:Calcium-Green-5N] complexes were monitored at the appropriatewavelength so as to follow changes in intracellular Ca²⁺ levelsthroughout the assay. The results are presented in FIG. 9.

Functional assays of the calcium uniporter (CaUP) were similarlyperformed on non-adherent L6 cells, as described above, with respiratoryinhibitors oligomycin and rotenone in order to observe the effects ofelimination of the membrane potential using this high throughput FLIPR.Oligomycin and rotenone were added to the MM, 160 uM CaCl₂ (see previousexample) to concentrations of 50 microgram/milliliter and 20 micromolar,respectively. Cells were prepared and permeabilized as described above,in wells of a 96-well CoStar 3603 microtiter plate. Controls wereprepared by the addition of 100 microliters of the permeabilizingsolution into wells lacking cells. The plate was placed in a FLIPR and11.1 microliters of the MM, 160 uM CaCl₂ with inhibitors were dispensedinto wells with L6 cells resulting in assay concentrations of 16micromolar Ca²⁺, 5 microgram/milliliter oligomycin, and 2 micromolarrotenone. MM, 160 uM CaCl₂ with no inhibitors was dispensed into thewells lacking cells and having an assay Ca²⁺ concentration of 16micromolar. After approximately 3 minutes a second dose was dispensedinto the wells. The emission wavelength was monitored and intracellularCa²⁺ levels observed throughout assay. The profiles for cells subjectedto addition of Ca²⁺ in the presence of the respiratory inhibitorsreflect the profiles of the wells that had Ca²⁺ and possessed no cellsillustrating that Ca²⁺ is not sequestered in the mitochondria. Whenmedia with no Ca²⁺ is added, the fluorescence changes only minimally(rows 3 and 4)] (FIG. 9).

D. Screening for Modulators of Ca²⁺ Uniporter Activity

A uniporter assay was performed in order to observe the inhibitoryeffect produced by the FCCP (carbonyl cyanidep-(trifluoromethoxy)phenyl-hydrazone; Sigma), a highly effectiveuncoupler of oxidative phosphorylation in mitochondria. FIGS. 11 and 12depict dose response experiments wherein FCCP is used as an uncoupler.Such experiments demonstrate the ability of the assays of the inventionto detect uncouplers as mitochondrial Ca²⁺ releasing agents andinhibitors of uniporter activity. For drug screening purposes the assayis designed to identify agents that inhibit uniporter activity, butwould also identify respiratory uncouplers. In some embodiments,however, it may be preferable to establish a system by which one couldscreen for molecules that specifically inhibit uniporter activity buthave little or no effect on mitochondrial effects.

The basic respiratory media was prepared as described in Table 3, andstored at 4° C., as were stock solutions of 0.5 molar glutamate andmalate (Sigma, St. Louis, Mo.; each brought to a pH of 7.0 with KOH) and1 molar MgCl₂ (Sigma). On the day of the assay a master mix solution(MM) was prepared by the addition of glutamate and malate from the stocksolutions to final concentrations of 5 millimolar each, 1 millimolarMgCl₂, and 0.5 micromolar Calcium-Green-5N (Molecular Probes, Eugene,Oreg.). The hexapotassium salt Calcium-Green-5N (Molecular Probes,Eugene, Oreg.) in the MM is a fluorescent dye that has low bindingaffinity to Ca²⁺. The cell membrane permeabilizing agent digitonin(Sigma, St. Louis, Mo.) was added to MM to a final concentration of0.007% to yield a solution designated MM-D. To this mixture CaCl₂ wasadded to a final concentration of 6 micromolar Ca²⁺ to yield a solutiondesignated MM-D, 6 uM Ca. A 10×Ca²⁺ stock solution was prepared byaddition of CaCl₂ to a final concentration of 200 micromolar in MM (nodigitonin), and 10× solutions of FCCP were also prepared in MM to giveconcentrations of 0, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 5.0, and 10.0micromolar.

HepG2 cells (human hepatocellular carcinoma, ATCC HB-8065) weretrypsinized from near-confluent flasks in growth media, Eagle's minimumessential medium (MEM) with non-essential amino acids, sodium pyruvateand Earle's balanced salt solution (BSS), 90%; FBS 10%. Cells werecentrifuged, growth media aspirated, and resuspended in thepermeabilizing solution MM-D, 6 uM CaCl₂. The concentration of cells wasadjusted to 1×10⁷ cells per milliliter in solution. One hundred (100)microliters of the cell suspension (1×10⁶ cells) were delivered to the96 well microtiter plate. The plate was placed in the FLIPR™ unit warmedto 37° C. essentially according to the manufacturer's instructions. An11.1 microliter aliquot of 10× stock solutions of MM-FCCP, havingdifferent concentrations of FCCP, were dispensed into each of 8 wellswith cells to yield FCCP assay concentrations of 0, 0.02, 0.04, 0.06,0.08, 0.1, 0.2, 0.5, and 1.0 micromolar in like fashion, 11.1microliters of the 10× stock solutions were added into 8 wells with nocells. FCCP stock solutions were robotically administered one minuteafter the beginning of the experiment.

A pulse of 12.3 microliters of the 10× stock CaCl₂ solution wasintroduced into each of the wells of the microtiter plates after aboutfour minutes. Emissions resulting from the binding of extramitochondrialCa²⁺ to the calcium indicator dye were monitored throughout the assay.The emissions from each well of the 12 different assays (8 wells pereach assay) were graphed as a function of time (FIG. 10). Row 4 in FIG.10 shows the results from 8 wells (A-H) when cells and calcium arepresent. In rows 5 to 12 of FIG. 10, the uncoupler FCCP was present inaddition to cells and calcium. The concentration of FCCP decreases asone progresses from Row 5 (1.0 uM) to Row 12 (0.02 uM), and the effectof the uncoupler on CaUP activity decreases in a corresponding manner.The results obtained when 0.02 or 0.04 uM FCCP was present (Rows 12 and11, respectively) are difficult to distinguish from the results obtainedwhen no FCCP was present (Row 4).

The average of the changes of fluorescence for the assays (1-12) versustime course where FCCP was added at one minute, and calcium was pulsedinto the assay media at approximately 4 minutes, were determined andgraphed as a function of time (FIG. 11). The fluorescence rises slightlyupon addition of FCCP at t=1 min.; without wishing to be bound bytheory, this probably occurs as a result of Ca²⁺ that was loaded intothe mitochondria when they were first resuspended in MM-D containing 6micromolar Ca²⁺.

The high-throughput uniporter assay protocol, carried out as describedabove for suspension cells, was used to determine the effect ofdifferent compounds on mitochondria and extramitochondrial Ca²⁺concentrations in the presence or absence of the Ca²⁺ uptake inhibitorof the endoplasmic reticulum, thapsigargin (Calbiochem). Agents examinedin this assay include calcium channel inhibitors, pro- andanti-oxidants, mitochondrial inhibitors and uncouplers, and the Ca-UPinhibitor Ru360. On the day of the assay, solutions of therespiratory/cell permeabilizing media containing digitonin withthapsigarin (MM-TD) and without thapsigarin (MM-D) were prepared aspreviously described. Calcium chloride was added to each to a finalconcentration of 6 micromolar Ca²⁺, resulting in MM-TD, 6 uM Ca andMM-D, 6 uM Ca. A MM solution was prepared with a 10×Ca²⁺ concentrationof 200 micromolar for the second Ca²⁺ pulse. The Ca²⁺-uniporterinhibitor Ru360 was prepared at a 10× concentration of 100 microlitersin MM. Ten times (10×) stock solutions of the other agents were preparedfor the assay by the addition of the compounds to final concentrationsof 10 micromolar into MM. The agents assayed included: inhibitors ofCa²⁺ channels (not including the uniporter), amiloride (ResearchBiochemicals International, RBI), nicardipine (RBI), nifedipine (RBI),nimodipine (RBI), trifluoroperazine (RBI), and verapamil (RBI); acalcineurin inhibitor/immunosuppressant drug, FK506 (tacrolimus)(Calbiochem); diltiazem (RBI), an effector of some Ca²⁺ channels; thepro-oxidant diamide (azodicarboxylic acid bis[dimethylamide]) (Sigma,St. Louis, Mo.), and the anti-oxidant N-acetyl cysteine (Sigma);mitochondria uncouplers of oxidative phosphorylation such as CCCP(carbonyl cyanide m-chlorophenyl-hydrazone) (Sigma) and respiratoryinhibitor such as rotenone (Sigma); the a Ca²⁺ ionophore, ionomycin(Calbiochem); dantrolene (RBI), an inhibitor of the endoplasmicreticulum Ca²⁺ channel; a K⁺-channel inhibitor, glyburide(glibenclamide) (Calbiochem); an inhibitor of mitochondrial electrontransport, PK-11195 (RBI); a peripheral benzodiaepine receptorantagonist; and the metabolite, creatine (Aldrich, Milwaukee, Wis.).Controls include water and 10% DMSO (Sigma) in MM.

Cells of the HepG2 cell line (hepatocellular carcinoma, ATCC HB-8065)were trypsinized from near-confluent flasks in growth media (Eagle's MEMwith non-essential amino acids, sodium pyruvate and Earle's BSS, 90%;FBS 10%). Cells were centrifuged, growth media was aspirated, and cellswere resuspended to a concentration of 1×10⁷ cells per milliliter inMM-TD, 6 uM Ca, or MM-D, 6 uM Ca. One hundred (100) microliters of thecell suspension (about 1×10⁶ cells) were delivered to wells of amicrotiter plate. The plate was placed in a FLIPR™ II (used in 96-wellformat) warmed to 37° C. An 11.1 microliter aliquot of the 10× stocksolutions with water or DMSO (controls), Ru360 (Ca-UP inhibitor), or thetest compounds, were dispensed into designated wells such that the assayconcentration of DMSO is 0.1%, Ru360 is 10 micromolar, and each of thetest compounds are at 1 micromolar concentrations. After an additionalthree minutes, a second addition of 12.3 microliters of the 10× CaCl₂(200 micromolar Ca²⁺), was dispensed into the wells of the microtiterplate resulting in assay concentrations of 20 micromolar Ca²(Extramitochondrial Ca²⁺ bound to the calcium indicator dye wasmonitored throughout and profiles of each assay; the results arepresented in FIG. 12, with all thapsigargin minus (thaps−) assays inwells corresponding to columns 1-6 and thapsigargin plus (thaps+) assaysin wells corresponding to columns 7-12.

Profiles of all assays are referred to by their location in themicrotiter plate, column vs row, of FIG. 13. Upon addition of testcompounds, most of the wells indicate little or no change in thefluorescence signal over the ensuing 3 minutes. This indicates little orno effect on the ability of the mitochondria to retain the 6 micromolarCa²⁺ that was accumulated upon permeabilization. The exception to thisis the effect of the uncoupler CCCP (A4, F2, A10, F8) and ionomycin(Ca²⁺ ionophore D6, G4, D12 and G10). There is also a slight indicationof Ca²⁺ release in response to atractyloside (B6, C4, B12, C10), aninducer of the permeability transition which induces Ca²⁺ releasethrough opening of a large conductance pore in the inner mitochondrialmembrane. Control assays having no affect on uniporter activity arewater (Al, G6, A7, G12), and DMSO (H1, H4, H7, H10). The Ca²⁺ ‘spike’reflects uptake of Ca²⁺ into the mitochondria and exhibit Ca²⁺-UPactivity. Similar profiles are observed for the non-uniporterinhibitors: amiloride (A2, D1, A7, D7); nicardipine (C2, G5, C8, G11);nifidipine (C3, E4, C9, E10); nimodipine (C5, F1, C11, F7);trifluoroperazine (D2, H6, D8, H12); verapamil (D4, G1, D10, G7); anddiltiazem (B3, H5, B9, H11), reflecting that they do not possessuniporter inhibitory activity at one micromolar. The uniporternon-inhibitory profile is also observed for: FK506 (B4, E6, B10, E12);diamide (B1, D5, B7, D11); N-acetylcysteine (C1, E2, C7, E8); dantrolene(A6, B2, A12, B8); glyburide (glibenclamide) (B5, F3, B11, F9); PK-11195(E1, H2, E7, H8); and creatine (F4, H3, F10, H9). Alternatively, theCa-UP inhibitor Ru360 shows no uptake of Ca²⁺ by the uniporter upon theCa²⁺ addition and is reflected in a plateau of Ca²⁺-calcium indicator inthe extramitochondrial medium (E5, F6, E11, F12). A similar profile isobserved for the respiratory inhibitor rotenone (C6, E3, C12, E9),probably indicating that upon addition of Ca²⁺ the ETC complex I isdisabled, the membrane potential is dissipated, and Ca²⁺ can no longerbe sequestered by the mitochondria. The uncoupler CCCP (A4, F2, A10, F8;release Ca²⁺ from mitochondria) and Ca²⁺ ionophore (D6, G4, D12, G10;release Ca²⁺ from cell stores) reflect profiles of agents that preventCa²⁺ uptake and retention by the mitochondria.

EXAMPLE 5 Detection of Modulation of Mitochondria PermeabilityTransition (MPT)

A. General Method

An assay was performed in order to observe the transition inductioneffect on MPT by the MPT effector, diamide (a thiol oxidizing agent andpro-oxidant). The assay was performed in a digitonin-permeabilized cellline of mixed cybrids, MixCon in the presence of Ca²⁺ and Ca-Green-5Nand monitored in an LS50B Perkin Elmer Spectrofluorimeter, but isadaptable to the FLIPR system. Initially, ethacrynic acid was added tothe MixCon cells in Dulbecco's modified Eagle's medium and 10% FBS, to afinal concentration of 100 micromolar and preincubated for 5 minutes toremove the ability of cells to deal with oxidative stress making cellssusceptible to the pro-oxidant. The cells were then centrifuged, growthmedia was aspirated, and cells resuspended, at 1.1×10⁷ cells/milliliter,in 2 milliliters of respiratory media containing digitonin (MM-D)whereupon the digitonin (dig) permeabilizes the cells. The suspensionwas then transferred to a spectrofluorimetric cuvette. The cuvette wasplaced in the fluorimeter and 0.3 microliters of a 254 millimolar CaCl₂stock was added to the cuvette (final CaCl₂ concentration of 40micromolar). After approximately three minutes diamide was added to thecuvette to a final concentration of 500 micromolar]. Withinapproximately 6 minutes, the mitochondria released the sequestered Ca²⁺supposedly via the effect of the pro-oxidant on the MPT. Addition of theuncoupler FCCP at an assay concentration of 0.1 micromolar characterizesno further release of Ca²⁺ from the mitochondria. Addition of CaCl₂ to40 micromolar in the assay resulted in increased fluorescence, thus theCa²⁺-sensitive dye was demonstrated not no to have been saturated withC²⁺ under assay conditions (FIG. 13).

To further scrutinize modulation of the MPT the same experiment wasperformed on MixCon cells, as described above, with the exception thatafter the cells were permeabilized 10 micromolar of the permeabiltytransition blocker, cyclosporin A (CsA), was added. Upon addition ofdiamide only a slight increase in fluorescence was observed. Supposedlythe CsA prevented the pro-oxidant from inducing the MPT as reflected bythe maintenance of the reduced level of Ca²⁺ in the extramitochondriamedium. Only upon addition of the uncoupler FCCP was Ca²⁺ released fromthe mitochondria as reflected by the elevated level of Ca²⁺ associatedwith the Ca²⁺ indicator (FIG. 13).

The transition induction effect on MPT by the MPT effector, diamide (athiol oxidizing agent and pro-oxidant) were performed with a second cellline, 293T. The 293T cells were suspended in 2 milliliters of growthmedia, DMEN and 10% FBS, with 100 micromolar of ethacrynic acid (1×10⁷cells/ml) and preincubated for 14 minutes to remove ability of cells todeal with oxidative stress making them susceptible to the pro-oxidant.The cells were centrifuged, media aspirated, resuspended in 2milliliters of respiratory media containing digitonin (MM-D), andtransferred into a cuvette. The digitonin (dig) in MM-TD permeabilizesthe cells. The cuvette was placed in a LS50B Perkin ElmerSpectrofluorimeter and 40 micromolar CaCl₂ in H₂O was added into thesuspension. After approximately three minutes 500 micromolar diamide wasdispensed into the cuvette. The mitochondria immediately began to slowlyrelease Ca²⁺ to the medium, and this rate substantially increased afterabout 7.5 minutes, reflecting the pro-oxidant's effect on the MPT.Addition of the uncoupler FCCP at an assay concentration of 0.1micromolar resulted in no increased level of fluorescence and gaveevidence that no additional Ca²⁺ was released from the mitochondria,therefore all Ca²⁺ had been freed into the cytosol by the effect ofdiamide.

B. Screening For Modulation of MPT Activity Using Adherent Cells orNon-Adherent Cells (Cuvettes or FLIPR)

A high-throughput assay with a FLIPR™ 384 was used to observe themodulation of MPT when permeabilized cells were subjected to themitochondria permeability transition inducer atractyloside (Sigma),permeability transition blockers bongkrekic acid (Calbiochem) andcyclosporin A (CsA), and the pro-oxidant, diamide. HepG2 cells(hepatocellular carcinoma, ATCC HB-8065), in suspension, were used todetermine the effect of these compounds on mitochondria Ca²⁺sequestration by monitoring extramitochondrial Ca²⁺ concentrations inthe presence or absence of the Ca²⁺ uptake inhibitor of the endoplasmicreticulum, thapsigargin (Calbiochem, San Diego, Calif.). On the day ofthe assay solutions of the respiratory media containing digitonin withthapsigarin (MM-TD) and without thapsigarin (MM-D) were prepared aspreviously described. Calcium chloride was added to each to a finalconcentration of 6 micromolar Ca²⁺, to yield MM-TD, 6 uM Ca, and MM-D, 6uM Ca. Ten times (10×) stock solutions were prepared for the assay bythe addition of each of the compounds to final concentrations of 10micromolar in MM. Ten times (10×) solutions of CaCl₂ was prepared in MMor MM-T to concentrations of 200 micromolar, for the second addition.

The HepG2 cells were trypsinized from near-confluent flasks in growthmedia (Eagle's MEM with non-essential amino acids, sodium pyruvate andEarle's BSS, 90%; FBS 10%). Cells were centrifuged, media was aspirated,and the cells were resuspended to 1×10⁷ cells per milliliter in MM-TD, 6uM Ca and MM-D, 6 uM Ca, whereby Ca²⁺ and the indicator dye enter thecells and Ca²⁺ becomes sequestered in mitochondria. One hundred (100)microliters of the cell suspension was delivered to each well of amicrotiter plate. The plate was placed in the FLIPR 384 warmed to 37° C.An 11.1 microliter volume of the 10× stock solutions with the testcompounds were dispensed into designated wells such that the assayconcentration are 1 micromolar. After a three minute interval, a Ca²⁺pulse of 20 micromolar was delivered by adding 12.3 microliters of the10× CaCl₂ solutions containing 200 micromolar Ca²⁺. Extraorganelle Ca²⁺bound to the calcium indicator dye was monitored throughout and profilesof each assay are presented in FIG. 13, with all thapsigargin minus(thaps−) assays in wells corresponding to columns 1-6 and thapsigarginplus (thaps+) assays in wells corresponding to columns 7-12.

Profiles of all assays are referred to by their location in microtiterplate, column vs row, of FIG. 13. Assay activity profiles of most of thecompounds, including inhibitors of permeability transition, bongkrekicacid and CsA show that upon addition of the Ca²⁺ pulse the suddenincrease in cytosolic Ca²⁺ is observed to decrease resulting in the‘spike’ indicative of normal mitochondria sequestration of Ca²⁺. Theassay profile of the permeability inducer atractyloside (B6, C4, B12 andC10) show initial uptake of Ca²⁺ into mitochondria with the slow releaseof the Ca²⁺ into the cytosol, probably through the MPT pore. Thepro-oxidant diamide, B1, D5, B7 and I1, reflects sequestering of Ca²⁺ bymitochondria after the Ca²⁺ pulse. This last result differs with that ofa similar assay in cuvettes (see above) where diamide had caused therelease of Ca²⁺ from mitochondria. The difference is attributed to thediamide concentrations of each experiment where is in cuvettes the assayconcentration of diamide was 0.5 millimolar, and in the FLIPR assay theuse of 1 micromolar diamide was too low to affect Ca²⁺ sequestration inmitochondria.

Hypothetically, to screen for inhibitors of the mitochondrial membranepermeability transition, the quantity of Ca²⁺ added to the cellsfollowing the addition of the test compound could be increased to alevel that induces spontaneous Ca²⁺ release. In the absence of any testcompound, a tracing indicative of the transition would appear as anincrease followed by an inability of the mitochondria to return theextramitochondrial Ca²⁺ concentration back down to baseline. Forinstance, the Ca²⁺ addition could be increased to approximately 400micromolar, which would result in mitochondrial Ca²⁺ uptake, followed bystimulation of the opening of this non-specific pore, allowing Ca²⁺ toefflux from the mitochondria. Addition of permeability transitioninhibitors such as bongkrekic acid (at 1 micromolar) or cyclosporin A(at 1 micromolar) would inhibit the transition and allow mitochondria tocontinue Ca²⁺ accumulation to bring the fluorescence level back tobaseline.

EXAMPLE 6 Detection of Modulation of Ca²⁺ Out of Mitochondria

General Method (Cuvettes or FLIPR)

This fluorescence-based assay could be modified to allow screening forcompounds that alter the mitochondrial buffer or set point (theconcentration of Ca²⁺ outside the mitochondria following activation ofuniporter and Ca²⁺ efflux activities). Mitochondrial efflux is normallymediated by either the Na⁺/Ca²⁺ exchanger or a Ca²⁺/H⁺ antiporter,depending upon the mitochondrial type. To carry out the assay, all thesame media and cell and/or mitochondrial preparations could remain aspreviously described with the exception that no Ca²⁺ would be added tothe permeabilization medium, and permeabilization medium would have noadded Ca²⁺. The cells could then be dispensed to the microtiter wells,and the first addition could be the addition of Ca²⁺ (approximately 4 to15 micromolar, but empirically determined to be well below aconcentration that induces the permeability transition) followed by0.1-5 μM ruthenium Red along with 30 micromolar NaCl to the medium (toenhance the rate of the Na⁺/Ca²⁺ exchanger if necessary). The additionof Na⁺ to cells in which the mitochondria exhibit Na⁺/Ca²⁺ exchangeactivity will result in a return of the fluorescence to a level abovethe original baseline. The test compound would be added afterapproximately 3 minutes, and the tracing would be evaluated for eitheran increase or decrease in the fluorescence compared to the equilibriumestablished prior to the addition of test compound. A decrease in thesteady-state level would be indicative of an efflux inhibitor. Anincrease in the steady-state level would be indicative of an effluxaccelerator. A second pulse of Ca²⁺ of the same magnitude as the firstaddition could be added. A compound that either speeds influx orinhibits efflux would result in a sharper Ca²⁺ peak and a more rapidreturn to the previous level of Ca²⁺. An accelerator of Ca²⁺ effluxwould broaden the spike and slow the return to baseline.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A method of identifying an agent that uncouples oxidativephosphorylation from ATP production, comprising: (a) contacting (i) abiological sample comprising a cell containing cytosol, a mitochondrionand a calcium indicator molecule, under conditions that permitmaintenance of mitochondrial membrane potential, and wherein the calciumindicator molecule is membrane permeable and capable of generating adetectable signal that is proportional to the level of calcium in thecytosol, with (ii) a calcium ionophore, under conditions and for a timesufficient to increase calcium levels within the cell; (b) detecting thesignal generated by the calcium indicator molecule at a plurality oftime points; (c) repeating steps (a) and (b) at least once; and (d)comparing (i) the signal generated by the calcium indicator molecule atone or more of said time points prior to and following at least one ofthe contacting steps in the absence of the candidate agent to (ii) thesignal generated by the calcium indicator molecule at one or more ofsaid time points prior to and following at least one of the contactingsteps in the presence of the candidate agent, wherein an increased levelof calcium in the cytosol at a time point prior to a contacting step inthe presence of the agent, compared to the level of calcium in thecytosol prior to a contacting step in the absence of the agent,indicates an agent that uncouples oxidative phosphorylation from ATPproduction.
 2. A method of identifying an agent that is a respiratoryinhibitor, comprising: (a) contacting (i) a biological sample comprisinga cell containing cytosol, a mitochondrion and a calcium indicatormolecule, under conditions that permit maintenance of mitochondrialmembrane potential, and wherein the calcium indicator molecule ismembrane permeable and capable of generating a detectable signal that isproportional to the level of calcium in the cytosol, with (ii) a calciumionophore, under conditions and for a time sufficient to increasecalcium levels within the cell; (b) detecting the signal generated bythe calcium indicator molecule at a plurality of time points; (c)repeating steps (a) and (b) at least once; and (d) comparing (i) thesignal generated by the calcium indicator molecule at one or more ofsaid time points prior to and following at least one of the contactingsteps in the absence of the candidate agent to (ii) the signal generatedby the calcium indicator molecule at one or more of said time pointsprior to and following at least one of the contacting steps in thepresence of the candidate agent, wherein an increased level of calciumin the cytosol at a time point prior to a contacting step in thepresence of the agent, compared to the level of calcium in the cytosolprior to a contacting step in the absence of the agent, indicates anagent that is a respiratory inhibitor.
 3. A method of identifying anagent that alters a mitochondrial calcium uniporter, comprising: (a)contacting (i) a biological sample comprising a cell containing cytosol,a mitochondrion and a calcium indicator molecule, under conditions thatpermit maintenance of mitochondrial membrane potential, and wherein thecalcium indicator molecule is membrane permeable and capable ofgenerating a detectable signal that is proportional to the level ofcalcium in the cytosol, with (ii) a calcium ionophore, under conditionsand for a time sufficient to increase calcium levels within the cell;(b) detecting the signal generated by the calcium indicator molecule ata plurality of time points; (c) repeating steps (a) and (b) at leastonce; and (d) comparing (i) the signal generated by the calciumindicator molecule at one or more of said time points prior to andfollowing at least one of the contacting steps in the absence of thecandidate agent to (ii) the signal generated by the calcium indicatormolecule at one or more of said time points prior to and following atleast one of the contacting steps in the presence of the candidateagent, wherein an increased level of calcium in the cytosol at a timepoint following a contacting step in the presence of the agent, comparedto the level of calcium in the cytosol following a contacting step inthe absence of the agent, indicates that the agent alters amitochondrial calcium uniporter.